Genome-Wide CRISPR-Cas9 Screen Identifies Sensitizers to LSD1 Inhibition in MLL-Translocated Human AML Cells

Lysine-specific demethylase (LSD1, also known as KDM1A) is an epigenetic regulator that has recently emerged as a potential therapeutic target in acute myeloid leukemia (AML). It is a flavin dependent monoamine oxidase which can demethylate monomethyl or dimethyl lysine 4 of histone H3. Pharmacological inhibition of LSD1 induces differentiation of blast cells in MLL-translocated AML and has shown significant promise in pre-clinical studies. With LSD1 inhibitors advancing through early-phase clinical trials, there is a strong pre-clinical rationale for the identification of genes whose protein products collaborate with LSD1 to retard differentiation in cancer and which could potentially be targeted in combination therapies for enhanced therapeutic benefit.

To identify potential drug-gene synthetic lethal interactions, we performed a genome wide loss-of-function CRISPR-Cas9 screen in human THP1 AML cells treated with a potent and selective tranylcypromine-derivative LSD1 inhibitor trans-N-((2-methoxypyridin-3-yl)methyl)-2-phenylcyclopropan-1-amine (OG86). THP1 cells exhibit a t(9;11) MLL gene rearrangement and display similar phenotypic and functional responses to those observed in primary MLL-translocated AML cells following LSD1 inhibition. The cells were transduced with lentiviral human CRISPR knockout (hGeCKOv2) library containing 122,411 sgRNAs targeting 19,050 protein coding genes and 1864 miRNA precursors in the human genome. The transduced cells were divided into two groups, treated with either DMSO or 250nM OG86 and maintained in culture for ~10 population doublings. To investigate sgRNA representation in the cell population harvested at different stages of the screen, sgRNA cassettes were PCR amplified from genomic DNA and deep sequenced; negatively selected genes were identified with the MAGeCK computational algorithm. After successful initial quality assessment of the screen, we next searched for genes selectively depleted in OG86-treated versus DMSO-treated THP1 cells in samples collected on Day 15 and Day 18. At a false discovery rate of 7.5% there were 10 expressed genes whose sgRNA representation was depleted at both time points. In particular, these included two genes coding for components of the MTOR signaling pathway: MTOR associated protein, LST8 homolog (MLST8) and Ras-related GTP-binding protein A (RRAGA). In the Day 18 comparison, an additional MTOR pathway gene LAMTOR2 scored among the ten most depleted. MLST8 is a core component of TORC1/TORC2 complex and RRAGA is involved in the activation of TORC1 by amino acids. Recruitment of both RAG proteins and TORC1 to lysosomal membranes in response to amino acids, and the consequent activation of TORC1 signalling, requires the trimeric Ragulator complex, of which LAMTOR2 is a member.Based on our screen, we hypothesized that THP1 AML cells exposed to pharmacologic inhibition of LSD1 exhibit increased sensitivity to concomitant inhibition of the amino acid sensing component of the TORC1 pathway.

Using genetic knockdown and pharmacological inhibition strategies, we then validated our screen hits in combination with LSD1 inhibition in targeting human AML cells. RNAi based knockdown of RRAGA, LAMTOR2 and MLST8 in combination with LSD1 inhibition were found to promote myeloid differentiation and reduce cell proliferation in THP1 cells. Interestingly the mTORC1 pathway inhibitor everolimus (RAD001) showed at least additive effect in combination with OG86 to decrease THP1 cell proliferation and promote immunophenotypic differentiation. Comparison of transcription changes in combined versus single treatment conditions by RNA-seq analysis further confirmed a more extensive and wide-ranging upregulation of a myeloid differentiation program upon concomitant inhibition of LSD1 and mTORC1 pathway. In vitro studies performed in primary patient AML cells gave similar results. Finally, in vivo studies using AML patient-derived xenograft mouse model confirmed that combination treatment promotes a strong myeloid differentiation program. In conclusion, we report here that inhibition of mTORC1 sensitizes human MLL-translocated AML cells to LSD1 inhibitor-mediated differentiation therefore highlighting a novel combination approach for evaluation in clinical trials.