The XPO7/Npat Axis Is a Potential Therapeutic Target for TP53-Mutated AML

Acute myeloid leukemia (AML) is a devastating disease with a long-term survival rate below 30%. While AML is a genetically heterogeneous disease, TP53 mutations are among the most powerful risk factors in AML, which underscores the critical need to devise a novel therapeutic strategy for TP53-mutated AML.

To identify genes/pathways whose loss is vulnerable to TP53 deficiency in AML cells, we performed genome-wide CRISPR-Cas9 screens using Trp53-knockout (KO) and wild-type (WT) mouse AML cells. To generate AML lines with a relatively "clean" genetic background, we established mouse AML lines harboring WT Trp53 with normal karyotype (Yamauchi et al. Cancer Cell, 2018). We then generated Trp53-KO AML lines using a single guide RNA (sgRNA) targeting Trp53. Genome-wide CRISPR-Cas9 dropout screens were performed using these lines to identify genes/pathways whose loss is vulnerable to TP53 deficiency.

We identified Xpo7, a putative nuclear/cytoplasmic transporter, as a factor necessary for the survival of Trp53-KO AML cells. sgRNAs targeting Xpo7 were enriched in Trp53-WT AML cells after the 16-day culture, while they were significantly depleted in Trp53-KO cells. Trp53-KO cells were vulnerable to Xpo7 depletion, while Xpo7 functioned as a Trp53-dependent tumor suppressor in Trp53-WT AML cells. As expected, CRISPR/Cas9-mediated Xpo7 depletion and shRNA-mediated Xpo7 knockdown significantly delayed cell cycle progression and suppressed cell growth only in Trp53-KO AML cells, but not in WT cells.

We next performed CRISPR-Cas9 saturation mutagenesis scan targeting all Xpo7 exons using Trp53-WT and -KO mouse AML cells. Strikingly, sgRNAs targeting Xpo7 coding regions were significantly enriched only in the Trp53-WT cells, namely in the presence of an MDM2 inhibitor. In stark contrast, the same sgRNAs which target coding exons including the C-terminal importin-β domain were mostly depleted in Trp53-KO AMLs.

Since Xpo7 presumably mediates the nuclear import and export of proteins, we hypothesized that Xpo7 promotes retention of Trp53 protein in the nucleus in Trp53-WT AML cells. As expected, Trp53 protein levels in the nucleus and mRNA levels of Trp53-targeting genes were significantly decreased upon Xpo7 depletion. Furthermore, Xpo7 depletion resulted in resistance to Selinexor, a XPO1 inhibitor that reportedly blocks Trp53 protein export from the nucleus. These data suggest that Xpo7 retains WT-Trp53 in the nucleus and functions as a Trp53-dependent tumor suppressor in Trp53-WT AML.

Next, to elucidate the mechanisms underlying the toxic effects observed in Trp53-KO AMLs upon Xpo7 depletion, we set out to identify proteins whose cytoplasmic/nuclear localization was potentially regulated by Xpo7 in Trp53-KO AML cells. We first performed IP-mass spectrometry using an anti-Xpo7 antibody to identify Xpo7 interacting proteins. Then, we obtained both cytoplasmic and nuclear protein extracts from both Xpo7-WT and -KO AML cells and identified proteins whose localization was significantly altered upon Xpo7 deletion by mass spectrometry. We found that Npat, an activator of histone transcription in the G1/S transition, interacted with Xpo7 and localized more in the cytoplasm upon Xpo7 depletion in Trp53-KO AMLs to a significant extent. Strikingly, Npat protein levels were robustly upregulated following Trp53 depletion, and Npat depletion significantly delayed the progression of the G1/S transition and proliferation in Trp53-KO AML cells.

Finally, to explore the functional significance of the XPO7/NPAT axis in human TP53-mutated AML cells, we assessed the correlation between XPO7 expression levels and AML subtypes and/or genetic background using publicly-available datasets. XPO7 and NPAT mRNA levels were significantly upregulated in TP53-mutated AMLs in the TCGA datasets (Ley et al. NEJM, 2013). Moreover, XPO7 and NPAT mRNAs levels were remarkably high in acute erythroid leukemia (AEL) cases, where TP53 mutations are frequently observed (Tyner et al. Nature, 2018 and Iacobucci et al. Nat Genet. 2019). In fact, shRNA-mediated XPO7 or NPAT knockdown significantly suppressed proliferation of HEL, a human AEL cell line harboring TP53 mutation, in vitro and in vivo.

In summary, we identified a synthetic lethal relationship between TP53 and XPO7. Our data suggest that XPO7/NPAT inactivation is a therapeutic vulnerability for TP53-mutated AML, such as AEL.

Ogawa:Pfaizer: Speakers Bureau; 2013-526957 (JP02): Patents & Royalties; The Mitsubishi foundation: Honoraria; Astellas: Speakers Bureau; Sysmex: Honoraria; Nanpu Hospital: Research Funding; DaiichiSankyo: Speakers Bureau; Astrazeneca: Speakers Bureau; The Chemo-Sero-Therapeutic Research Institute: Speakers Bureau; Clinical Research Support Center Kyushu: Research Funding; 2013-096582 (JP01): Patents & Royalties; Esai Pharmatheutical: Consultancy; ASAHI Genomics: Current equity holder in publicly-traded company; Novartis: Honoraria, Speakers Bureau; Kirin/Chugai: Speakers Bureau; Chordia Threapeutics: Consultancy, Current equity holder in publicly-traded company, Research Funding; 2015-239547: Patents & Royalties; PCT/JP2014/062112 (WO01): Patents & Royalties; 15/353395 (US03): Patents & Royalties; 62/187386 (US01): Patents & Royalties; 2014-191287: Patents & Royalties; MSD: Speakers Bureau; Otsuka Pharmatheutical: Research Funding.