CKS1 inhibition reveals vulnerabilities in leukemic stem cells with concomitant protection of healthy hematopoietic stem cells

Acute myeloid leukemia (AML) is an aggressive hematological disorder comprising a hierarchy of quiescent leukemic stem cells (LSCs) and proliferating blasts with limited self-renewal ability. AML has a dismal prognosis, with extremely low two-year survival rates in the poorest cytogenetic risk patients, primarily due to the failure of intensive chemotherapy protocols to deplete LSCs, and the significant toxicity towards healthy hematopoietic cells. Whilst much work has been done to identify genetic and epigenetic vulnerabilities in AML LSCs, little is known about protein homeostasis in drug resistance and relapse. By targeting the proteostatic regulator CKS1, we demonstrate a dual role for CKS1-dependent protein degradation in reducing AML blasts in vivo, and importantly depleting LSCs, whilst inhibition of CKS1 has the opposite effect on normal hematopoiesis, protecting normal hematopoietic stem cells from chemotherapeutic toxicity. Together these findings demonstrate CKS1-dependent proteostasis is a key vulnerability in malignant stem cell biology.

buffer to a final volume of 300 L and subjected to cysteine alkylation using sequential incubation with 10 mM dithiothreitol (DDT) and 16.6 mM iodoacetamide (IAM) for 1 h and 30 min, respectively, at 25 °C with agitation. Trypsin beads (50% slurry of TLCKtrypsin; Thermo-Fisher Scientific; Cat. #20230) were equilibrated with 3 washes with 20 mM HEPES (pH 8.0), the urea concentration in the protein suspensions was reduced to 2 M by the addition of 900 L of 20 mM HEPES (pH 8.0), 100 L of equilibrated trypsin beads were added and samples were incubated overnight at 37°C. Trypsin beads were removed by centrifugation (2000 xg at 5°C for 5 min) and the resulting peptide solutions were desalted using carbon C18 spin tips (Glygen; Cat. # TT2MC18). Briefly, spin tips were activated twice with 200 L of Elution Solution (70% ACN, 0.1% TFA) and equilibrated twice with 200 L of Wash Solution (1% ACN, 0.1% TFA). Samples were loaded and spin tips were washed twice with 200 L of Wash Solution. Peptides were eluted into fresh tubes from the spin tips with 4 times with 50 l of Elution Solution. In each of the desalting steps, spin tips were centrifuged at 1,500xg at 5C for 3 min. Finally, samples were dried in a SpeedVac and peptide pellets were stored at −80°C.
For mass spectrometry identification and quantification of proteins, samples were run twice in a LC-MS/MS platform. Briefly, peptide pellets were resuspended in 100 L and 20 L of reconstitution buffer (20 fmol/L enolase in 3% ACN, 0.1% TFA) for THP-1 and CD34 + samples, respectively. Then, 2 L were loaded onto an LC-MS/MS system consisting of a Dionex UltiMate 3000 RSLC coupled to a Q Exactive Plus Orbitrap Mass Spectrometer (Thermo Fisher Scientific) through an EASY-Spray source (Cat. # ES081, Thermo Fisher Scientific). Mobile phases for the chromatographic separation of the peptides consisted in Solvent A (3% ACN: 0.1% FA) and Solvent B (99.9% ACN; 0.1% FA). Peptides were loaded in a micro-precolumn (Acclaim PepMap 100 C18 LC; Cat. # 160454, Thermo Fisher Scientific) and separated in an analytical column (Acclaim PepMap 100 C18 LC; Cat. # 164569, Thermo Fisher Scientific) using a gradient running from 3% to 23% over 120 min. The UPLC system delivered a flow of 2 L/min (loading) and 300 nL/min (gradient elution). Drug sensitivity and resistance testing (DSRT) Single drug DSRT was performed as described previously (52). In brief, compounds, each with 7 different concentrations, were pre-plated using an acoustic liquid handling Echo 550 (Labcyte) to 384-well plates. Primary AML cells were suspended in conditioned medium (RPMI 1640 supplemented with 10% fetal bovine serum, 2mM Lglutamine, penicillin-100U/ml, streptomucing-100ug/ml and 12.5% conditioned medium from HS-5 human bone marrow stromal cells), DNase I treated for 4h (Promega), filtered through a 70m cell strainer (Thermo Fisher Scientific) to remove possible cell clumps, and viable cells were counted. Pre-plated compounds in each 384-well plate were dissolved in 5ul of conditioned medium using a MultiDrop Combi peristaltic dispenser (Thermo Fisher Scientific) and shaken for 5 minutes to dissolve the compounds. AML cells were plated at 5,000 cells/well in 20ul, leading to a final volume of 25ul/well. Plates were gently shaken for 5 minutes to mix the cells with the compounds and incubated for 72 hours at 37ºC, 5% CO2.
Cell viability was measured using the CellTiter-Glo assay (Promega) with a PHERAstar microplate reader (BMG-labtech). Data was normalised to negative (DMSO only) and positive control wells (100uM benzethonium chloride) and dose response curves calculated.
Ex vivo drug sensitivity of AML cells to the tested drugs was calculated using a drug sensitivity score (DSS), a modified form of the area under the inhibition curve calculation that integrates multiple dose response parameters for each of the tested drugs, as previously described (53).

Intestinal crypt analyses
Tamoxifen (Sigma, #T5648) was dissolved in ethanol to 300 mg/ml and further diluted in sunflower seed oil (Sigma #S5007) to a final concentration of 30 mg/ml. To induce recombination, 6-14 weeks old Lgr5 tm1(cre/ERT2)Cle mice were given one dose of tamoxifen (150 ug/g body weight) via oral gavage. After 24h, chemotherapy was administered as described above. After seven days the animals were culled, the intestines harvested and fixed in 10% neutral buffered formalin for 24h and subsequently transferred to 70% ethanol. After embedding and sectioning, the slides

Colony forming units
For resident mouse hematopoietic cell response to 5-FU', CKS1i, DA and DAC, colony forming ability was assessed in methylcellulose (StemCell Technologies M3434-GF). 10 4 mCD45 + cells were sorted from PDX mice at the indicated points and seeded in methylcellulose and scored to colony forming units after 7 days. Cultures were dissolved in PBS, counted and 10 4 cells were re-seeded for passage 2 and passage 3.

Viability assays
Relative cell viability was assessed by % reduction O2 in culture wells using the Alamar blue cell viability reagent (Life Technologies). Cells were seeded in 96 well plates at 2x10 5 cells/ml and the indicated dose of drugs were added on top and incubated for 48 hours. Alamar blue reagent was added on top of cells, and cells were incubated for another 4 hours under the same conditions (37ºC, 5% CO2). Plates were read on a spectramax plate reader (Biostars) at 570nm and 600nm and % reduction O2 was calculated as per the manufacturer's instructions.
Flow Cytometry, apoptosis and cell cycle assays Flow cytometry analysis was performed using a BD Fortessa flow cytometer (BD biosciences). Cells were prepared by washing in PBS + 1% FBS three times before staining in the same media with the indicated cell surface antibodies (resources table) for 1 hour at 4C. For apoptosis assays, cells were incubated with annexin V binding buffer in addition to the washing media (BD biosciences), washed three times in PBS + 1% FBS + 1x annexin V binding buffer and incubated with 0.1g/ml DAPI prior to flow cytometry analysis. For cell cycle analysis, cells were washed three times in PBS + 1% FBS and fixed in BD fix/perm buffer (BD biosciences) for 20 minutes at room temperature. Cells were washed three times in BD perm/wash buffer + 0.1% Triton X-100 (BD biosciences) and incubated with intracellular antibodies, such as anti-Ki67, for 4 hours at 4C. Cells were washed three times in BD perm/wash buffer and 0.5 g/ml DAPI was added for 15 minutes prior to analysis. For all flow cytometry, cells were initially identified based on forward and side scatter.

RNA extraction, reverse transcription and real time quantitative PCR (RT-qPCR)
Total RNA was isolated from patient samples after thawing, density centrifugation and T-cell depletion, using a RNeasy mini kit (Qiagen). Resulting RNA was reverse transcribed to produce cDNA using the Superscript III reverse transcriptase kit (Thermo Fisher Scientific) with oligoDT20 primers (Sigma Aldrich). RT-qPCR experiments were performed with an ABI-7500 FAST Thermal Cycler (Applied Biosystems) using SYBR Green (Thermo Fisher Scientific). RNA abundance was quantified by the Comparative CT method with two independent control genes (GAPDH and B-ACTIN, GAPDH presented). The CT values used for each patient sample were the result of three technical triplicates. Primers are described in the resources table.
RAC1/RHOA G-LISA assay Analysis of RAC1/RHOA-GTP abundance was carried out using the RAC1/RHOA G-LISA assay as per the manufacturer's instructions (Cytoskeleton inc.). Control and CKS1i treated AML cells were lysed on ice with the provided lysis buffer for 10 minutes and centrifuged at 10,000g, 4C, for 5 minutes. Protein was quantified and normalized with precision red protein reagent. Lysate, lysis buffer only or control protein was incubated with G-LISA wells at 4C for 30 minutes with agitation. Wells were washed three times with wash buffer and primary antibody incubation was carried out at room temperature for 45 minutes with agitation. Wells were washed three times with wash buffer and secondary antibody incubation was carried out at room temperature for a further 45 minutes with agitation. HRP detection reagent was added to each well and