The Histone Acetyltransferase MOZ (KAT6A) Is a Molecular Dependency and Therapeutic Target in NUP98-Rearranged Acute Myeloid Leukemia
Authors: Michmerhuizen Nicole L , Heikamp Emily , Umeda Masayuki , Iacobucci Ilaria , Arthur Bright , Di Giacomo Danika , Hiltenbrand Ryan , Radko-Juettner Sandi , Portola Pablo , Claquin Aurelie , Chandra Bappaditya , Baggett David W , Huang Hongling , Zhou Peipei , Long Lingyun , Shi Hao , Sun Yu , Papachristou Evangelia K , Chilamakuri Chandra Sekhar Reddy , Vitorino Francisca N. de L. , Gongora Joanna M , D'Santos Clive S , Garcia Benjamin A , Kriwacki Richard W , Chi Hongbo , Kentsis Alex , Armstrong Scott A , Klco Jeffery M , Mullighan Charles G. 
Published: December 2024

NUP98 fusion oncoproteins(FOs) are a hallmark of high-risk leukemia and are present in approximately 5% of pediatric and 3% of adult acute myeloid leukemia (AML). NUP98 fusion oncoproteins involve the N-terminal, intrinsically disordered region of NUP98 and the C-terminal portion of one of over 30 fusion partners. Approximately one-third of fusion partners have DNA-binding homeodomains, and many others have domains involved in gene regulation. Outcomes in children with NUP98-rearranged (NUP98-r) AML are poor, with relapse rates of nearly 70%.
We and others recently demonstrated the importance of NUP98 FO-mediated liquid-liquid phase separation in NUP98-r leukemic transformation (Cancer Discov 2022;12:1152, Nature 2021;595:591), but the composition of NUP98 FO-associated condensates and role of FO-interacting proteins in leukemogenesis are incompletely understood. To identify FO interacting proteins, we performed rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) for HA-tagged wildtype NUP98 and eight NUP98 FOs (NUP98::HOXA9, PMX1, KDM5A, NSD1, JADE2, LNP1, RAP1GDS1, SETBP1) in transfected HEK293T cells. We identified that many known NUP98 FO interactors (e.g. XPO1, RAE1, KMT2A, Menin) were shared across all tested NUP98 FOs, and we observed novel FO interactors including MOZ and other histone acetyltransferase (HAT) complex members. Imaging of HEK293T cells expressing GFP-tagged NUP98::LNP1 demonstrated colocalization of FO with MOZ and associated histone acetylation marks within FO condensates.
To investigate the functional importance of chromatin remodeling complexes including HATs for NUP98::KDM5A-driven leukemogenesis, we performed an in vivo CRISPR/Cas9 screen with a guide RNA library targeting 337 epigenetic genes. Our results suggested that BRPF1, a chromatin writer of histone H3 acetylation marks that associates with MOZ and other HAT complexes,is a molecular dependency in NUP98::KDM5A AML. Competitive co-culture of Nup98::Kdm5a;Vav-Cre;Cas9 cells expressing control or HAT complex-targeting gRNAs further validated that inactivation of Brpf1 and a subset of other MOZ/MORF HAT complex members (Moz, Hbo1, Brd1 or Meaf6) decreased cell fitness.
We next tested efficacy of pharmacologic inhibition of MOZ in NUP98-r leukemia models. The commercially available MOZ/HBO1 inhibitor PF9363 resulted in reduced viability and myeloid differentiation in mouse and human models of NUP98-r leukemia with multiple fusion partners. In hematopoietic stem and progenitor cells from Nup98::Kdm5a;Vav-Cre mice, PF9363 treatment resulted in global loss of H3K14ac and H3K23ac as determined by mass spectrometry, and reduction in H3K9ac at NUP98 FO target genes by CUT&RUN.
In vivo, PF9363 (3 mg/kg daily) reduced leukemic burden and/or prolonged survival in 1 NUP98::HOXA13 and 2 NUP98::NSD1 PDX models. As disease progressed upon treatment cessation, we tested PF9363 in combination with Menin inhibitor SNDX-5613, since Menin inhibitors have shown activity in NUP98-r leukemia (Blood 2022;139:894) and are currently being evaluated in clinical trials. In mouse NUP98 FO leukemia cells, combined in vitro treatment with PF9363 and SNDX-5613 led to decreased cell viability and heightened myeloid differentiation as compared to either agent alone. Single-agent treatment with PF9363 or SNDX-5613 resulted in loss of the NUP98 FO and BRPF1 from chromatin at select FO target genes, including Meis1. Combination treatment decreased both expression of select Hox transcription factors and NUP98 FO chromatin occupancy at HoxA/B cluster genes, which was not observed with either PF9363 or SNDX-5613 monotherapy.
Finally, we tested the combination of PF9363 and SNDX-5613 in vivo using NUP98-r PDX models that are sensitive or resistant to Menin inhibition. In a Menin inhibitor sensitive NUP98::KDM5A PDX, combined treatment with PF9363 and SNDX-5613 resulted in increased expression of myeloid differentiation markers compared to SNDX-5613 alone. In a NUP98::NSD1 PDX that is resistant to Menin inhibition (Blood 2022;139:894), PF9363 monotherapy extended median latency by 21%. In combination with SNDX-5613, PF9363 increased median latency by 50% compared to vehicle and by 18% compared to PF9363 alone.
In summary, our studies show that MOZ is a therapeutic vulnerability in NUP98 FO-driven AML and that MOZ inhibitor treatment may improve responses to Menin inhibition.