Ethics approval

All research and methods were performed in accordance with the relevant guidelines and regulations. All in vivo procedures were conducted in accordance with and approved by the Institutional Animal Care and Use Committee of Washington University (no. 20-0463). The maximal disease burden denoted by the ethics committee/institutional review board (e.g., moribund and sluggish appearance, hunched and with ruffled coats, harboring visible lesions) was not exceeded. Animals were humanely euthanized upon immobility or appearance with signs of distress.

Cell culture

HEL (ATCC), SET2 (ATCC), UKE-1 (Coriell Institute), MOLM-13 (DSMZ), MEG-01 (ATCC) cells were cultured in RPMI 1640 ATCC modification (ThermoFisher, Waltham, MA). MV4-11 (ATCC) cells were cultured in IMDM. THP-1 (ATCC) cells were cultured in RPMI 1640 supplemented with 0.05 mM beta-mercaptoethanol. Ba/F3 (DSMZ) cells were cultured in RPMI 1640 supplemented with 1 ng/mL murine IL-3, while Ba/F3 transduced with FLT3-ITD and FLT3-D835Y were cultured in the absence of murine IL-3. HEK293T (ATCC) cells were cultured in DMEM. All cell lines were maintained at 37 °C and 5% CO2, grown in media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, and regularly tested for mycoplasma.

Compounds, immunoblot and flow cytometry antibodies

PMD-026 was provided by Phoenix Molecular Designs. Ruxolitinib, fedratinib, midostaurin, and quizartinib were purchased from Selleck Chemicals (Houston, TX). BI-D1870, MG-132, and SJB3-019A were purchased from MedChemExpress (Monmouth Junction, NJ). Murine IL-3 was purchased from Peprotech (Rocky Hill, NJ). Cycloheximide was purchased from Sigma-Aldrich (St. Louis, MO).

Immunoblotting antibodies were used as follows: pRB S795 (#9301), cyclin D1 (92G2, #2978), RSK1 (D6D5, #8408), p90RSK T359/S363 (#9344), pS6 S235/236 (D57.2.2E, #4858), pFLT3 Y591 (54H1, #3466), FLT3 (8F2, #3462), USP1 (D37B4, #8033), HA (C29F4, #3724), Ubiquitin (P37; #58395), and K48-linkage Specific Polyubiquitin (D9D5, #8081) from Cell Signaling Technology (Danvers, MA). HSP90 (sc-13119) and WDR48 (E-4, sc-514473) from Santa Cruz Biotechnology (Dallas, TX). Flow cytometry antibodies were used as follows: anti-mouse CD45.1-PE (110708) and anti-mouse CD45.2-APC (109814) from BioLegend (San Diego, CA).

Plasmid constructs and transduction

Lentiviral and retroviral transduction with low multiplicity of infection (MOI) was performed following protocol described at http://www.broadinstitute.org/rnai/public/resources/protocols. Transduced cells were selected in puromycin for 2–3 days and plated for downstream assays immediately after selection.

Individual shRNA vectors used were from the Mission TRC library (Sigma) provided by the McGill Platform for Cellular Perturbation (MPCP) of Rosalind and Morris Goodman Cancer Research Centre at McGill University as follows: control vector pLKO.5, shRPS6KA1 #1 (TRCN0000001386), shRPS6KA1 #2 (TRCN0000001388). The following shRNAs were purchased from Sigma: shUSP1 #1 (TRCN0000004043), shUSP1 #2 (TRCN0000004046). FLT3-ITD-MSCV-IRES-GFP and FLT3-D835Y-MSCV-IRES-GFP were gifts from Timothy Ley (Addgene plasmid # 184779; http://n2t.net/addgene:184779; RRID:Addgene_184779, Addgene plasmid # 184780; http://n2t.net/addgene:184780; RRID:Addgene_184780); pLenti puro HA-Ubiquitin was a gift from Melina Fan (Addgene plasmid # 74218; http://n2t.net/addgene:74218; RRID:Addgene_74218).

Cell viability assays

Cell lines were seeded into 96-well plates upon gene perturbation or treated with indicated doses of inhibitors and cultured for 72–96 h. At endpoint, resazurin viability reagent was added and absorbance was read in a BioTek microplate reader.

Immunoblotting

Cells were collected and lysed with Laemmli Sample Buffer (BioRad, Hercules, CA) with beta-mercaptoethanol and heated at 95 °C. Samples were processed with NuPAGE Novex Gel Electrophoresis Systems (ThermoFisher) followed by standard immunoblotting procedure.

Immunoprecipitation (IP) and ubiquitin assay

Co-IPs were performed using the Pierce Classic Magnetic IP/Co-IP Kit (Thermofisher). In brief, cells were pelleted, washed with PBS and lysed with IP lysis/wash buffer (pH 7.4, 0.025 M Tris, 0.15 M NaCl, 0.001 M EDTA, 1% NP40, and 5% glycerol). Cells were then centrifuged, an input fraction was collected, and the supernatant was incubated with 1 μg of RSK1 primary antibody or IgG control (Cell Signaling #2729) for 2 h at room temperature to form the immune complex. Immune complexes were then incubated with protein A/G magnetic beads, eluted, and collected following heating under reducing conditions.

For the ubiquitin assays, 293 T cells were transfected with HA-Ubiquitin and FLT3-ITD. Following 36 h, cells were treated with PMD-026 and MG-132 for 16 h. IP was then performed following protocol above with 1 ug of HA antibody. Ba/F3 cells were transduced with FLT3-ITD and IP was performed following 16 h treatment with PMD-026 and using 1 ug of FLT3 antibody. Two sets of SJB3-019A treatment experiments were performed: Ba/F3 cells transduced with 1) FLT3-ITD, and 2) FLT3-ITD and HA-Ub. IP was performed following 2 h treatment with SJB3-019A and using 1 μg of HA or FLT3 antibody.

Cycloheximide (CHX) assay

Transduced MV4-11 cells were treated for the indicated hours in the presence of cycloheximide before collection and immunoblotting. Line plots showing protein normalization were performed through densitometry with ImageJ (NIH) where bands were normalized by the internal loading control and to the 0 CHX treatment condition within each group.

Flow cytometry and apoptosis assay

Cells were stained in live cell buffer and run on the BD FACSCanto II Cell Analyzer utilizing unstained controls, and downstream analysis was performed with FlowJo (Ashland, OR). Cells were collected and stained per the PE Annexin V Apoptosis Detection Kit I (BD Biosciences). In brief, post-treatment, cells were washed with PBS and resuspended in binding buffer, incubated with PE-Annexin V, and stained with 7-AAD viability staining solution. Cells were then analyzed by flow cytometry, in which AnnexinV + /7-AAD+ cells were identified as necrotic, AnnexinV + /7-AAD− as apoptotic, and AnnexinV − /7-AAD− as healthy.

Sequencing and downstream analysesRNA-sequencing (RNA-seq)

Cells were treated in duplicate and RNA was extracted using the RNeasy Mini Kit (Qiagen) with Dnase treatment. cDNA synthesis was performed using PolyA selection. Samples were then indexed, pooled, and sequenced by Illumina NovaSeq 6000. Basecalls and demultiplexing were performed with Illumina’s bcl2fastq software and a custom python demultiplexing program with a maximum of one mismatch in the indexing read. RNA-seq reads were then aligned to the Ensembl release 76 primary assembly with STAR version 2.5.1a [21]. TMM normalization size factors in EdgeR [22] were calculated to adjust for samples for differences in library size. Ribosomal genes and genes not expressed in the smallest group size minus one samples greater than one count-per-million were excluded from further analysis. The TMM size factors and the matrix of counts were then imported into the R/Bioconductor package Limma [23]. Additional analysis was performed using DEseq2 [24], using GSEA [25] software with the Hallmark gene set, or pathway analysis at https://www.gsea-msigdb.org/.

Animal modelsMV4-11 xenograft

1 × 106 cells were engrafted into sub-lethally irradiated (200 cGy) female 6–9 week-old NSGS mice by tail-vein injection. Two weeks post-transplantation animals were randomized and vehicle or 100 mg/kg PMD-026 was administered by oral gavage twice a day for 28 days. Human CD45+ cells in the peripheral blood were measured every week and bone marrow at endpoint by flow cytometry. Measurements and assessments were performed blindly.

Vav-Cre Flt3 ITDTet2 KO model

1 × 106 Vav-Cre Vav-Cre Flt3ITDTet2KO CD45.2 cells were injected into the tail veins of sub-lethally irradiated CD45.1 female mice. Three weeks post-transplantation animals were randomized, PMD-026 was administered by oral gavage (100 mg/kg/BID) for 28 days. Hematological parameters were analyzed by flow cytometry and Hemavet. Measurements and assessments were performed blindly.

Multivariate and Kaplan–Meier survival analysis

For multivariate analysis, patient clinical information was retrieved from TCGA LAML [4] and BeatAML [5] from vizome.org. Between these two cohorts, overlapping clinical features included sex, race, and ELN 2017 risk, and samples with these factors, survival data, and expression data of FLT3, RPS6KA1, and USP1 were pooled together. Multivariate analysis was performed, where expression was stratified by greater or lesser than the median gene expression. Kaplan-Meier analysis was performed on the pooled TCGA and BeatAML cohorts and patients were stratified into four groups based on USP1 and RPS6KA1 gene expression (singular high or low, double high, or double low, where high and low represent comparison to the median expression).

Public databasesDepMAP

Gene effect scores (CERES) [26] from CRISPR (Avana) public 21Q1 were accessed from the DepMap portal by the Broad Institute.

BeatAML2

OHSU BeatAML2 [14] clinical and expression data were obtained from http://vizome.org.

TCGA LAML

TCGA LAML mutational and clinical data were accessed from cBioPortal [27].

Leucegene cohort

Survival data were obtained from data.leucegene.iric.ca.

scRNA-seq of gilteritinib-treated AML patients (GSE199333)

Feature files and clinical information were assessed from GSE199333 and source publication [15]. Data were processed following scRNA-seq pipelines as previously described [8]. Following population annotation, myeloid cells were subsetted for downstream gene expression analyses. No new code was generated. R scripts used in this study are available from corresponding author upon request.

Other statistical analysis and figures

Statistical analyses were performed using GraphPad Prism 9 (San Diego, CA) and R software. All measurements were taken from distinct samples. All relevant assays were performed independently at least 3 times. Figures were generated with GraphPad Prism 9, and R software.