In acute myeloid leukemia (AML), TP53 mutations are the most prognostically unfavorable genetic alterations, as they confer resistance to various therapeutic agents and form relapsing clones, underscoring the critical need to devise a novel therapeutic strategy for TP53-mutated AML. To identify genes or 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. We employed two distinct KO screen libraries, through which we identified Xpo7, a putative nuclear/cytoplasmic transporter, as a common factor necessary for Trp53-KO AML survival. Interestingly, Trp53-KO cells were vulnerable to Xpo7 depletion, while Xpo7 functioned as a Trp53-dependent tumor suppressor in Trp53-WT AML cells. Further probing this phenomenon, we performed a CRISPR-Cas9 saturation mutagenesis scan on all Xpo7 exons. Strikingly, sgRNAs targeting Xpo7 coding regions were significantly enriched only in the Trp53-WT AMLs. In stark contrast, these same sgRNAs were mostly depleted in Trp53-KO AMLs. To determine the role of Xpo7 depletion on the expansion of Trp53 deficient clones, we prepared AML cells containing a small fraction of Trp53-KO cells both in the Xpo7-WT and KO backgrounds and assessed the proportion over time in vitro or in vivo. Remarkably, the expansion of Trp53-KO clones, noticeable in the Xpo7-WT setting, was significantly reduced under the Xpo7-KO background, prolonging the mice's survival. These data suggest that Xpo7 depletion effectively impedes Trp53-KO clone expansion. Since Xpo7 is presumed to mediate the nuclear import and export of proteins, we hypothesized that Xpo7 contributes to retaining Trp53 protein within the nucleus in Trp53-WT AMLs. As expected, nuclear Trp53 protein and mRNA levels of Trp53-targeting genes decreased significantly upon Xpo7 depletion. These data suggest that Xpo7 retains WT-Trp53 in the nucleus and functions as a Trp53-dependent tumor suppressor in Trp53-WT AMLs. Next, to elucidate the underlying mechanisms causing the toxic effects in Trp53-KO AMLs upon Xpo7 depletion, we set out to identify proteins whose localization was potentially regulated by Xpo7 in the Trp53-KO state. Employing mass spectrometry on an immunoprecipitate using anti-Xpo7 and cytoplasmic/nuclear protein extracts from both Xpo7-WT and KO AML cells, we found that Npat, an activator of histone transcription in the G1/S transition, interacted with Xpo7 and showed a significant shift towards cytoplasmic localization upon Xpo7 depletion in Trp53-KO AMLs. Strikingly, Npat protein levels displayed a notable upregulation by Trp53 depletion in AML cells. To evaluate the relevance of the XPO7/NPAT axis in human TP53-mutated AML cells, we examined the correlation between XPO7 and NPAT expression levels and AML subtypes and genetic background using publicly available datasets. XPO7 and NPAT mRNA levels were significantly upregulated in TP53-mutated AMLs in the TCGA datasets. 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; Iacobucci et al. Nat Genet. 2019). The Xpo7 and Npat protein expression evaluation using primary AML samples corroborated these findings, revealing overexpression in TP53-mutated AML cases. Finally, we assessed the therapeutic effect of Npat depletion in Trp53-deficient AML cells, using the dTAG system to induce Npat protein degradation by knock-in FKBP12 F36V into Npat loci. dTAG v-1 treatment immediately degraded endogenous Npat protein and led to delayed G1/S transition progression. Moreover, Npat degradation induced DNA damage, particularly in the presence of a WEE1 inhibitor or etoposide, and subsequent apoptosis. Transplantation of these AML cells into immunocompromised mice followed by dTAGv1 treatment for one week demonstrated suppressing leukemia progression in vivo. Furthermore, shRNA-mediated XPO7 or NPAT knockdown significantly suppressed the 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. We unveiled the novel regulatory mechanism of protein localization by XPO7, depending on TP53 mutational state. Our data suggest that XPO7/NPAT inactivation is a therapeutic vulnerability for TP53-mutated AML.
Read full abstract
Jul 11, 2024
Amir Masoud Firouzabadi + 3
Open Access
