Abstract 4030: Efficacy of novel combination therapy against AML cells with mutant NPM1

Abstract

Abstract Cytoplasmic dislocation and loss of nucleolar localization of the chaperone protein NPM1c occurs in 30% of AML following a frame shift C-terminal mutation in exon 12 of NPM1 (creating NPM1c), which contributes to differentiation arrest, growth and survival of AML stem progenitor cells. In present studies, we determined that knock out (KO) of mutant (mt) NPM1 (NPM1c) in the AML OCI-AML3, utilizing targeted gRNA (compared to control) and CRISPR-Cas9, induced growth arrest, morphologic differentiation and loss of viability of the KO cells over 5 to 9 days. This was associated with depletion of protein levels of NPM1c, c-Myc, and MEIS1, but upregulation of PU.1, RUNX1, CD11b, p21 and NOXA levels. ChIP-Seq analysis with H3K27Ac antibody 5d after KO demonstrated reduced peak density at the super-enhancers of MYC, MEIS1, and HOXA9. RNA-Seq analysis showed negative enrichment of HALLMARK gene sets of mRNA expression of MEIS1/HOXA9, MYC targets, ribosome/translation and cell cycle. Compared to OCI-AML3 with NPM1c, OCI-AML3 with NPM1 KO exhibited abrogation of the Menin inhibitor SNDX-50469-induced differentiation and apoptosis. Additionally, NPM1 KO also significantly reduced sensitivity of OCI-AML3 cells to apoptosis induced by targeted agents previously shown to demonstrate efficacy against AML cells with NPM1c, including KPT-330 (XPO1 inhibitor), homoharringtonine (protein translation inhibitor), and anti-AML chemotherapeutic agents, including daunorubicin, cytarabine and etoposide. However, NPM1 KO sensitized OCI-AML3 cells to ATRA (all trans retinoic acid)-induced differentiation and loss of viability, which was associated with marked induction of p21 and CD11b. Utilizing RNA-Seq signature of NPM1c KO and interrogating the LINCS1000-CMap data set of gene expression signatures, we determined that among the top expression mimickers were several pan-HDAC inhibitors and a WEE1 kinase inhibitor. Treatment with adavosertib (WEE1 inhibitor, MK-1775) or romidepsin (HDAC inhibitor) dose-dependently induced apoptosis in OCI-AML3 cells, as well as in 5 samples of patient-derived (PD) AML cells with NPM1c. Moreover, co-treatment with adavosertib and romidepsin synergistically induced lethality in OCI-AML3 and PD AML cells with NPM1c. This was associated with marked reduction in protein expressions of c-Myc, c-Myb, MEIS1 and CDK4/6, but upregulation of TP53 and p21 levels. These findings highlight that presence of NPM1c mechanistically regulates the sensitivity of AML cells to Menin inhibitor, ATRA and anti-AML chemotherapeutic agents. They also identify the novel non-chemotherapy combination of pan-HDAC inhibitor and WEE1 inhibitor for its potential synergistic activity against AML with NPM1c. Citation Format: Christopher P. Mill, Warren C. Fiskus, John A. Davis, Courtney D. DiNardo, Christine Birdwell, Qi Jin, Koichi Takahashi, Joseph D. Khoury, Kapil N. Bhalla. Efficacy of novel combination therapy against AML cells with mutant NPM1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4030.