Mitochondrial Respiration Regulates GPX4 Inhibition-Induced…

Mitochondrial Respiration Regulates GPX4 Inhibition-Induced Ferroptosis in Acute Myeloid Leukemia Hiroki Akiyama , Ran Zhao, Yuki Nishida, Lauren B Ostermann, Po Yee Mak, Edward Ayoub, Sujan Piya, Bing Z Carter, Michael Andreeff and Jo Ishizawa Section of Molecular Hematology and Therapy, Department of Leukemia, University of Texas MD Anderson Cancer Center

Results

Background

Ferroptosis, a form of non-apoptotic cell death regulated by iron-dependent lipid peroxidation, has drawn extensive attention as potential anti- cancer strategy. However, it remains to be explored in hematologic malignancies. We here investigate the molecular mechanisms of ferroptosis in acute myeloid leukemia (AML) and its therapeutic potential with co-targeting of mitochondrial respiration. Hypothesis Ferroptosis pathway is a therapeutic vulnerability in AML  Oxidative stress and iron overload in AML cells  Induction of cell death that bypasses apoptosis resistance

Anti-leukemia effect of GPX4 inhibition is independent of TP53 mutational status

ClpP-mediated degradation of mitochondrial respiratory complex proteins 6 sensitizes AML cells to ferroptosis

GPX4 is a potential therapeutic target in AML with prognostic relevance

TP53 modulations in MOLM13 (CRISPR-mediated gene editing) or Kasumi-1 (knockdown of mutant p53) do not change the ML210 sensitivity

cervix breast pancreas upper_aerodigestive urinary_tract skin gastric colorectal esophagus colorectal

Cell death

0 20 40 60 80 100

Dox; ClpP activation ML210 Combo

ONC201 Dox; shGPX4 Combo

-Low GPX4 -High GPX4

Cell death

0 20 40 60 80 100

Kasumi-1-

MOLM13-TP53-

shC shTP53

WT KO

bile_duct fibroblast peripheral_nervous_system soft_tissue AML bone thyroid ALL kidney rhabdomyosarcoma ovary mesothelioma multiple_myeloma liver central_nervous_system lymphoma uterus lung prostate

C.I. = 0.57

R175H R248Q

- -

1.25 0.25

2.5 0.5

5.0 1.0

- -

0.25 0.1

0.5 0.2

0.75 0.3

1.0 0.4

Dox (ug/ml) ML210 (uM)

ONC201 (uM) Dox (ug/ml)

Primary samples n = 11

ONC201 degrade respiratory complex proteins (published 6 )

- - 0 20 40 60 80 100 Cell death (representative primary sample) ML210 ONC201 Combo ML210 (uM) ONC201 (uM) 5.0 2.5 10 5.0 20 10

1.0

n= 53 ea. Logrank p = 0.013

ML210 (uM)

GPX4 inhibition induces lipid-associated oxidative stress and ultrastructural alterations in mitochondria

0.5

-2.0 -1.5 -1.0 -0.5 0.0

High GPX4 mRNA expression associates with shorter survival in AML patients (TCGA)

0.0

Dependency score AML cell lines are one of the cell lines highly dependent (low dependency score) on GPX4 among those on DepMap (shinyDepMap)

Combination of ClpP hyperactivation (ONC201 or dox-induced CLPP-Y118A hyperactivated mutant) and GPX4 inhibition (ML210 or dox-induced knockdown) in AML cell lines and primary AML progenitor (CD34 + CD38 - ) cells

Mitochondrial superoxide

GPX4 was one of the top sensitization hits on CRISPR screening for ClpP hyperactivation

GPX4 inhibition induces ferroptosis in AML cells GPX4 knockdown by doxycyclie-inducible shRNA induces lipid peroxidation followed by cell death in AML cells

Materials and Methods

Summary

Public datasets: shinyDepMap 1 , GEPIA 2 , CRISPR screening for NALM6 cells treated with ClpP agonists 3 Cells; Parental AML cell lines, OCI AML3-shGPX4, MOLM13-TP53mut 4 , Kasumi1-shTP53, HL60-Rho0 5 , OCI-AML3-CLPP-Y118A 6 , Primary AML patient samples Reagents; GPX4 inhibitor; ML210, ClpP agonist; ONC201, lipophilic antioxidants; Liproxstatin-1 (Lip1) and a-Tocopherol (aToc), iron chelator; deferoxamine (DFX), mitochondrial antioxidants; MitoQ and MitoQH2 Flow cytometry; AnnexinV/DAPI cell death assay, C11 BODIPY581/591 and MitoPerOx lipid peroxidation assays, MitoSOX red mitochondrial superoxide indicator, Western blot Transmission electron microscopy

Acknowledgement  GPX4 inhibition induces ferroptosis in AML cells  Mitochondrial respiration protects AML cells from GPX4- mediated ferroptosis  ClpP-mediated degradation of mitochondrial respiratory complex proteins and GPX4 inhibition synergistically exerts anti-leukemia effects  Studies are in progress to determine the molecular mechanisms and the in vivo efficacy of the combination Relay For Life My Oncology Dream Award from Japan Cancer Society (HA) The Mochida Memorial Foundation for Medical and Pharmaceutical Research (HA) The Research Fellowship from The Uehara Memorial Foundation (HA) Institutional Research Grant at MD Anderson Cancer Center (JI) Leukemia Research Foundation (JI) Paul & Mary Haas Chair (MA) NCI Cancer Center Support Grant P30CA16672 (Flow Cytometry & Cellular Imaging Core Facility and High Resolution Electron Microscopy Facility, MDACC)

GPX4 inhibition induces  mitochondrial superoxide blocked by lipophilic antioxidants  mitochondrial lipid peroxidation

Small mitochondria with condensed membrane in ML210-treated cells

Mitochondrial respiration protects AML cells from ferroptosis

Cell death

GPX4 inhibition by ML210 induces lipid peroxidation and cell death in AML cell lines. The pharmacologic effects are blocked by lipophilic antioxidants and the iron chelator DFX.

Specific cell death

(-) MitoQ

0 20 40 60 80 100

Specific cell death

OCI-AML3 OCI-AML2 MOLM-13 MOLM-14 MV-4-11 HL-60 U-937 KG-1 NB-4 Kasumi-1

0 20 40 60 80 100

Rho0 Rho-WT

References

- Dox

shGPX4

1. Shimada, K., et al. eLife 2021;10:e57116 2. Tang, Z., et al. Nucleic Acids Research 2017;45, 98–102.

ML210 (uM)

ML210

Respiration-deficient HL60- Rho0 cells are more sensitive to GPX4 inhibition

MitoQ/QH2 block the anti-leukemia effects of GPX4 inhibition

3. Jacques, S., et al. Genetics 2020;214:1103–1120. 4. Boettcher, S., et al. Science 2019;365(6453):599–604. 5. Pelicano, H., et al. Journal of Biological Chemistry 2003;278(39):37832–37839. 6. Ishizawa, J., et al. Cancer Cell 2019;35(5):721-737.e9.

THP-1

0.1

ML210 (uM)

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