Paics, a De Novo Purine Synthetic Enzyme, Is a Novel Target for AML Therapy

Progress has been made in deciphering molecular mechanisms underlying AML pathogenesis due in part to near-complete understanding of AML genomes. However, AML is yet a devastating disease with a long-term survival rate of less than 30%, underscoring an urgent need for the development of additional therapeutic modalities. To identify novel targets for AML therapy, we performed genome-wide CRISPR-Cas9 dropout screens employing two mouse AML cell lines (CALM/AF10 and MLL/AF9), followed by a second screen in vivo. These two cell lines, which we established, harbor wild-type (WT) Trp53with normal karyotype, enabling us to interpret screening results more easily due to a "clean" genetic background. We then validated our findings using human AML cell lines and patient-derived xenograft (PDX) models (Yamauchi et al. Cancer Cell 2018).

In the current study, we assessed the screening results furtherusing MAGeCK MLE program (Li et al. Genome Biology 2015)and the DepMap (https://depmap.org/), a publicly available genome-wide CRISPR-Cas9 screen datasets of cancer cell lines including 15 human AML cell lines. We show that PAICS (Phosphoribosylaminoimidazole carboxylase), which encodes an enzyme involved in de novo purine biosynthesis, is a molecule essential for AML cell survival. MRT252040, a newly-developed PAICS inhibitor (PAICSi), efficiently killed AML cell lines with different genetic backgrounds and significantly prolonged survival of AML PDX models. Furthermore, we investigated the mechanism action of PAICSi employing additional functional screens: CRISPR-Cas9 mutagenesis scan of all Paicscoding exons and a genome-wide CRISPR/Cas9 dropout screen in the presence of PAICSi.

Read counts for each Paics-targeted single-guide RNA (sgRNA) significantly decreased in vitro (AML cell lines) and in vivo (mouse AML model). We then assessed the functional significance of PAICS inhibition in AML cell survival via shRNA-mediated PAICSknockdown. AML cells expressing PAICS shRNA exhibited a proliferative disadvantage compared to non-transduced cells or those expressing scrambled shRNA, indicating a toxic effect of PAICS depletion in AML cells. We next asked whether inhibition of PAICS enzymatic activity affects AML cell proliferation and/or apoptosis using PAICSi. We assessed AML growth rate, cell cycle status and apoptosis and found that inhibition of PAICS enzymatic activity delays AML cell proliferation via inducing cell cycle arrest and apoptosis. As expected, CRISPR-Cas9 mutagenesis scan showed that sgRNAs targeting the exonic regions relevant to PAICS enzymatic activity were significantly decreased after the 16-day incubation.

We next performed genome-wide CRISPR-Cas9 screens in the presence of PAICSi, followed by second screens using a small-scale sgRNA library containing 8-10 sgRNAs per candidate gene.We identified genes potentially involved in PAICSi resistance as well as those whose loss are synthetic lethal to PAICS inhibition. X-box-binding protein 1 (Xbp1) was among the top hits in the genes relevant to PAICSi resistance genes, and sgRNAs targeting Xbp1significantly enriched in the presence of PAICSi. In contrast, sgRNAs targeting Slc43a3or Hprt, both of which are implicated in the purine salvage pathway, were significantly dropped-out, indicating that PAICSi-mediated anti-leukemia effects can be enhanced upon concurrentinhibition of the purine salvage pathway.

Finally, we explored potential anti-leukemia effects of PAICSi in vivo using AML PDX models established from two human AML lines. PAICSi exhibited anti-leukemic activity, as evidenced by the lower leukemia burden in peripheral blood and bone marrow of PAICSi-treated mice. They survived significantly longer than vehicle-treated mice, indicative of therapeutic efficacy of PAICSimonotherapy against AML in vivo.

In summary, we identified PAICS as an essential gene for AML cell survival. We propose that pharmacological targeting of the de-novo purine synthesis pathway via PAICSi is a potential therapeutic strategy for AML therapy.