Abstract
Mammalian cells have developed sophisticated defense mechanisms to counteract a wide variety of stresses to which they are continuously exposed. These adaptive mechanisms are rewired in cancers, such as acute myeloid leukemia (AML), to permit oncogenic transformation (Luo J et al, Cell, 2009). Using an MLL-AF9 syngeneic mouse model, we performed a pooled in vivo shRNA screen intended to identify novel stress response vulnerabilities in AML. p97 / VCP, an AAA-ATPase protein chaperone known to be involved in protein homeostasis and ER stress, was identified as a top candidate.
We first validated AML cell dependency on VCP in vivo in the MLL-AF9 model and in vitro in a panel of human AML cell lines (n=16) and primary patient samples (n=5), using VCP-directed shRNA, overexpression of a VCP dominant negative mutant or a highly selective small-molecule inhibitor of VCP, NMS-873 (Magnaghi P et al., Nat Chem Biol, 2013). The on target effect of NMS-873 in an AML context was validated using a VCP mutant (A530T), which confers resistance to VCP inhibition.
We next sought to dissect the molecular mechanism by which VCP is essential to AML cell survival and proliferation. Unexpectedly, we determined that targeting VCP did not impair AML cell viability through alteration of the "proteotoxic stress" response (no accumulation of polyubiquitinilated proteins, no consistent change in proteasomal enzymatic activities and no correlation of NMS-873 sensitivity to bortezomib sensitivity in a panel of 16 AML cell lines). Using a VCP dominant negative mutant unable to translocate into the nucleus, we demonstrated that the inhibition of the nuclear, but not the cytoplasmic, fraction of VCP was sufficient to abrogate leukemic cell viability.
To define new potential interacting partners of VCP that could explain its pro-leukemogenic function, we used an immunoprecipitation-mass spectrometry approach in the MV4-11 AML cell line and established by pathway overlapping analysis a significant enrichment of DNA repair pathways among the VCP protein interactome network in AML cells. Further analysis confirmed DNA damage induction through gH2AX accumulation in response to VCP inhibition and a marked reduction of homologous recombination (HR) measured using flow cytometry-based reporter assays. In further support of VCP's role in HR signaling, VCP inhibition blocked activation of the serine/threonine kinase ATM and its direct downstream targets (BRCA1, SMC1, and KAP1) in response to DNA damage induction by etoposide in AML. Indeed, the pattern of sensitivity of a panel of 16 AML cell lines and 16 primary patient samples to an ATM inhibitor, KU-55933, was highly correlated with sensitivity to the VCP inhibitor (Spearman score 0.78 and 0.72, respectively).
In conclusion, we identified and validated VCP as a druggable dependency in AML and dissected the mechanistic underpinnings of VCP's role in HR orchestration through activation of ATM.
Disclosures
DeAngelo: Amgen: Consultancy, Research Funding; Pfizer Inc.: Consultancy, Honoraria, Research Funding; Shire: Honoraria; ARIAD: Consultancy, Research Funding; Blueprint Medicines: Honoraria, Research Funding; Celgene: Research Funding; BMS: Consultancy; Takeda Pharmaceuticals U.S.A., Inc.: Honoraria; Incyte: Consultancy, Honoraria; Glycomimetics: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding; Immunogen: Honoraria, Research Funding. Stone: Janssen: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees, Research Funding; Ono: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Seattle genetics: Membership on an entity's Board of Directors or advisory committees; Fujifilm: Membership on an entity's Board of Directors or advisory committees; Argenix: Other: DSMB; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz: Membership on an entity's Board of Directors or advisory committees; Orsenix: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Cornerstone: Membership on an entity's Board of Directors or advisory committees; Otsuka: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Sumitomo: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Arog: Membership on an entity's Board of Directors or advisory committees, Research Funding; Actinium: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees. Hermine: Hybrigenics: Research Funding; Novartis: Research Funding; Celgene: Research Funding; INatherys: Equity Ownership, Research Funding; AB Science: Equity Ownership, Honoraria, Patents & Royalties, Research Funding. Stegmaier: Novartis: Consultancy, Research Funding.
The zinc finger transcription factor EGR-1 impedes interleukin-1-inducible tumor growth arrest.