DOI: 10.5061/dryad.bzkh189g0
title: RNA sequencing of AML cells treated with milademetan, selinexor and the combination of milademetan and selinexor

description: The tumor suppressor TP53 is frequently inactivated in cancers in a
 mutation-independent manner and is reactivated by inhibiting its negative
 regulators. We here co-target MDM2 with the nuclear exporter XPO1 to
 maximize transcriptional activity of p53. MDM2/XPO1 inhibition accumulated
 nuclear p53 and elicited a 25- to 60-fold increase of its transcriptional
 targets. TP53 regulates MYC, and MDM2/XPO1 inhibition disrupted
 the c-MYC-regulated transcriptome, resulting in synergistic induction of
 apoptosis in acute myeloid leukemia (AML). Surprisingly,
 venetoclax-resistant AMLs express high levels of c-MYC and are vulnerable
 to MDM2/XPO1 inhibition in vivo. However, AML cells persisting after
 MDM2/XPO1 inhibition exhibit a quiescence- and stress response-associated
 phenotype. Venetoclax overcomes that resistance, as shown by single-cell
 mass cytometry. The triple inhibition of MDM2, XPO1, and BCL2 was highly
 effective against venetoclax-resistant AML in vivo. Our results propose a
 novel, highly translatable therapeutic approach leveraging p53
 reactivation to overcome non-genetic, stress-adapted venetoclax
 resistance.
description: AML cell lines OCI-AML3, MOLM-13 TP53 wild-type and MOLM-13 TP53
 p.R248Q cells were treated with milademetan, selinexor, and the
 combination of milademetan and selinexor for 12 hours. RNA samples were
 collected from those cells and subjected to RNA sequencing.
 RNA sequencing OCI-AML3, MOLM-13 TP53
 wild-type, and R248Q mutant cells were treated with DMSO, milademetan,
 selinexor, or the combination of Mil + Sel for 12 hours. Cells were
 harvested and total RNA was extracted using QIAGEN RNeasy extraction kit
 from each sample (N = 3 for each treatment, N = 12 in total) according to
 the manufacturer’s protocol. Barcoded,
 Illumina-compatible, strand-specific total RNA libraries were prepared
 using the TruSeq Stranded Total RNA Sample Preparation Kit (Illumina, San
 Diego, CA). Briefly, 1mg of DNase I treated total RNA was depleted of
 cytoplasmic and mitochondrial ribosomal RNA (rRNA) using Ribo-Zero Gold
 (Illumina). After purification, the RNA was fragmented using divalent
 cations and double-stranded cDNA was synthesized using random primers. The
 ends of the resulting double-stranded cDNA fragments were repaired,
 50-phosphorylated, 3’-A tailed and Illumina-specific indexed adapters were
 ligated. The products were purified and enriched by 11 cycles of PCR to
 create the final cDNA library. The libraries were quantified using the
 Qubit dsDNA HS Assay Kit (ThermoFisher, Waltham, MA) and assessed for size
 distribution using the Fragment Analyzer (Advanced Analytical, Ankeny,
 IA), then multiplexed 4 libraries per pool. Library
 pools were quantified by qPCR and sequenced, one pool per lane, on the
 Illumina NovaSeq6000 sequencer using the 150 bp paired-end
 format.  Differential gene expression profiling and
 pathway analyses Gene expression was quantified using
 the Kallisto/sleuth pipeline. Read quantification was performed with
 Kallisto (v. 0.44.0), a pseudo-alignment-based method to quantify RNA
 abundance at the transcript level in transcripts per million (TPM) counts.
 Kallisto quant was utilized with the number of bootstraps set to 100 using
 ENSEMBL cDNA transcripts (Human assembly hg19, release GRC38.14) for
 indexing. Hierarchical clustering and principal component analysis (PCA)
 of the samples were performed. Sleuth v0.30.0 was used to measure the
 abundance of the transcripts as transcripts per million (TPM) with
 covariates for treatment conditions and downstream differential gene
 expression (DGE) to leverage the bootstrap estimates of Kallisto and to
 output model-based, gene-level normalized TPM matrix. The abundance of the
 genes was calculated as the sum of the TPMs mapped to a given gene. When
 using the sleuth preparation, gene isoforms were aggregated with a target
 map file derived from the Refseq hg19 transcriptome. For each gene, both
 the likelihood ratio test and Wald test were performed on the condition
 parameter to obtain their respective FDR-corrected p-values. Significantly
 altered genes were those passing the two tests at a cutoff of false
 discovery rate (FDR) &lt; 0.1. The plot_transcript_heatmap function in
 the Sleuth package was utilized to visualize the cluster analysis.
 Enhanced Volcano R package was used to generate the volcano plots (Blighe,
 K.; Rana, S.; Lewis, M. EnhancedVolcano: Publication-Ready Volcano Plots
 with Enhanced Colouring and Labeling. 2018. R Package Version 1.3.5.
 Available online: https://github.com/kevinblighe/EnhancedVolcano), which is a visual tool for displaying differentially expressed genes (DEGs) among overall gene expression levels. Beta score (log2) of -1 and 1 (0.5 fold and 2 folds), and q value of 0.05 were used as threshold lines for the magnitude of change in gene expression and significance, respectively. HALLMARK gene sets were used for pathway enrichment and functional classification of differentially expressed genes. 
description: # RNA sequencing of AML cells treated with milademetan, selinexor and the
 combination of milademetan and selinexor ## Description of the data and
 file structure There are 144 zipped fastq files (gz) and one metadata
 file. Please refer to the metadata file to annotate the data of interest.
 Please read the methods in the description of the datasets to understand
 and reproduce the data in the manuscript. ## Code/Software used in the
 study Software packages to analyze our datasets are described in the
 method section.