Pre-clinical activity of combined LSD1 and mTORC1 inhibition in MLL-translocated acute myeloid leukaemia

The histone demethylase lysine-specific demethylase 1 (LSD1 or KDM1A) has emerged as a candidate therapeutic target in acute myeloid leukaemia (AML); tranylcypromine-derivative inhibitors induce loss of clonogenic activity and promote differentiation, in particular in the MLL-translocated molecular subtype of AML. In AML, the use of drugs in combination often delivers superior clinical activity. To identify genes and cellular pathways that collaborate with LSD1 to maintain the leukaemic phenotype, and which could be targeted by combination therapies, we performed a genome-wide CRISPR-Cas9 dropout screen. We identified multiple components of the amino acid sensing arm of mTORC1 signalling—RRAGA, MLST8, WDR24 and LAMTOR2—as cellular sensitizers to LSD1 inhibition. Knockdown of mTORC1 components, or mTORC1 pharmacologic inhibition, in combination with LSD1 inhibition enhanced differentiation in both cell line and primary cell settings, in vitro and in vivo, and substantially reduced the frequency of clonogenic primary human AML cells in a modelled minimal residual disease setting. Synergistic upregulation of a set of transcription factor genes associated with terminal monocytic lineage differentiation was observed. Thus, dual mTORC1 and LSD1 inhibition represents a candidate combination approach for enhanced differentiation in MLL-translocated AML which could be evaluated in early phase clinical trials.

Enrichment of gene sets positively and negatively correlated with leukemia stem cell potential in murine MLL leukemias (Table S4). CD11b

Human tissue, cell lines & cell culture
Human THP1 cells were from DMSZ (Braunschweig, Germany) and were cultured under standard conditions (5% CO 2 , 37°C) in RPMI1640 supplemented with 10% FBS, 10U/mL penicillin, 10µg/mL streptomycin and 2mM L-glutamine (all from Life Technologies, Carlsbad, CA), or methylcellulose (H4320, Stem Cell Technologies, Vancouver, Canada). Cells were verified for authenticity by STR analysis and confirmed to be free from mycoplasma contamination. Culture densities were 5x10 4 -5x10 5 for cells in liquid culture and the starting density was 10 3 /ml in semi-solid culture. Colonies were enumerated after 5-7 days.
Cryopreserved leukaemic blast cells collected from the bone marrow or blood of patients at presentation were thawed and co-cultured on MS5 stromal cells, as described, S1 in α-MEM medium supplemented with 12.5% heat-inactivated FBS, 12.5% heat-inactivated horse serum, 2mM L- were readily separated from stromal cells (adhesive clumps) through disruption of the stromal layer by pipetting and then filtering the whole through a 75µm filter basket (Partec, Gorlitz, Germany). OG-86 was synthesised in house, as described. 4 The water-soluble compound OG-98 was obtained from Oryzon Genomics S.A. S2,S3 Resorufin (alamarBlue) was from Invitrogen (Carlsbad, CA).

Murine experiments
Cryopreserved leukaemic blast cells from a patient with a t(10;11) MLL gene rearrangement (BB518) were thawed and subjected to human CD3 + immunomagnetic bead depletion (#130-050-101, Miltenyi Biotec, Bergisch Gladbach, Germany) to remove residual T-cells using the POSSELD program of an (sufficient for ten population doublings). Cells were divided between two groups of ten T225 flasks; each flask was seeded at 2×10 5 cells/mL in a total volume of 120mL. Cells were split every three days and medium and compound replaced. At the end of the experiment cells were frozen at -80°C for subsequent genomic DNA isolation using a QIAamp Blood Maxi kit (Qiagen, Hilden, Germany) as per manufacturer's instructions.
The sgRNA cassettes were amplified from genomic DNA and sequenced as detailed in the Supplemental Information. Sequencing reads were de-multiplexed using forward primer barcodes.
Reads were trimmed using cutadapt (v1.7.1). The 20bp sgRNA sequences were then aligned to the GeCKO library using Bowtie (v1.0.1) allowing for up to one mismatched base. Mapped sgRNAs were filtered according to the following criteria before quantification: (i) any sgRNAs with other targets in the genome that match exactly or differ by only one base were discarded and (ii) for each biological sample, any sgRNA with only a single read was removed. Normalised abundance for each sgRNA in a given sample was calculated as follows: Normalised reads per sgRNA = (reads per sgRNA/total reads for all sgRNAs in the sample)×10 6 To identify significantly depleted sgRNAs and genes in the screen we used the Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK) method.

Verification of uniform representation of CRISPR library
To verify uniform representation of sgRNAs a two-step PCR was performed in which PCR1 amplifies the lentiviral sequence containing the 20bp sgRNA cassette and PCR2 attaches Illumina sequencing adapters and barcodes. In PCR#1, 50ng of each half library was amplified using V2 adaptor primers (http://genome-engineering.org/gecko/) using Phusion High Fidelity master mix (Thermo). The thermocycling conditions were: 98C for 3min, 15 cycles of (98C for 20s, 60C for 20s, 72C for 30s) and 72C for 7min. For each half library there were two PCR#1 replicates. 5uL of each #PCR1 product was run in 1.5% TAE-agarose ethidium bromide gel to verify amplicon length (~300bp). For the PCR#2 reaction, which is carried out to add appropriate Illumina sequencing adapters to the PCR#1 product, 5uL of each PCR#1 product was used as template. Unique barcoded forward and reverse primer combination were used for each PCR#2 reaction as described. 12 Thermocycling conditions and cycle numbers were the same as for PCR#1. 5uL of PCR#2 product was run on a 1.5% TAE-agarose ethidium bromide gel to verify amplicon length (~350bp). All PCR#2 products were then pooled and gel purified from 2% TAE-agarose ethidium bromide gel using a QiaQuick kit, as per manufacturer's instructions (Qiagen, Hilden, Germany). The pooled library was then quantified by Qubit fluorometric quantitation (Thermo Fisher, Waltham, MA) and sequenced with 10% PhiX using a MiSeq System (150 cycles, v3 chemistry) (Illumina, San Diego, CA).
The sgRNA cassettes recovered from lentivirally-infected cells were amplified using PCR#1 and PCR#2 reactions as described above, but with some modifications. To preserve full library complexity and representation during sequencing, 244µg genomic DNA (~300-fold coverage) was used in each PCR#1 reaction per sample. For each PCR#1, we used 5ug gDNA using V2 adaptor primers and Phusion High Fidelity master mix (Thermo Fisher package. Pre-ranked gene set enrichment analysis was performed with GSEA v2.0.14 software from www.broadinstitute.org/gsea7. Genes were rank ordered according to log2 fold change in expression.

Cytospin analyses
2-5x10 4 cells were suspended in 150μl PBS and, through centrifugation at 600xg for 5 minutes, were spun onto a microscope glass slide and left to air dry. Cells were fixed by incubation in methanol for 10 minutes and then stained by May-Grunwald (Sigma Aldrich; diluted 1:1 with Sorenson's Buffer (33.3mM KH 2 PO 4 , 64.75mM Na 2 HPO 4 , pH 6.8)) staining for 20 minutes and then Giemsa (Sigma Aldrich; 10x diluted with Sorenson's Buffer) staining for 30 minutes. Stained slides were washed under running tap water and left in Sorenson's buffer for five minutes prior to one final brief wash with tap water. Slides were left to air dry before cells were permanently mounted with a coverslip and DPX neutral mounting media (VWR, Radnor, PA). Images were obtained using a Leica SCN400 histology scanner (Leica, Solms, Germany).
alamarBlue assay 2500 cells were plated into each well of a 96-well plate and incubated with inhibitors or DMSO vehicle for 5 days. 20µl alamarBlue was added to the cells and incubated for 4h at 37°C. Resorufin fluorescence was measured using a fluorescence-based plate reader (POLARstar Omega, BMG Labtech, Aylesbury, UK).

Amino acid depletion assay
Amino acid-free RPMI medium powder (R8999-04A, US Biological, Salem, MA) was complemented with 2g/L sodium bicarbonate and 0.8g/L sodium phosphate, dissolved in water, adjusted to pH7.4 and sterile filtered. Complete RPMI containing a 1x concentrated solution of amino acids was obtained by complementing amino acid-free RPMI medium with RPMI 1640 amino acids solution (R7131, Sigma Aldrich), adjusted to pH7.4 and filtered. 10U/mL penicillin, 10µg/mL streptomycin, 2mM L-glutamine were added shortly before use. Amino acid depletion media were obtained by supplementing the amino acid free RPMI with individual amino acids (all from Sigma Aldrich) as follows: L-Arginine (0.2mg/ml), L-Asparagine (0.05mg/ml), L-Aspartic acid (0.02mg/ml), L-