Page 184 - Haematologica - Vol. 105 n. 6 - June 2020

C. Bonolo de Campos et al.
Other novel compounds targeting the PIKfyve kinase, APY0201, YM201636, AS2677131, AS2795440, and MF4, have followed.13-16 Here we report that APY0201 and other PIKfyve inhibitors are effective inhibitors of MM cell viability in in vitro and ex vivo models of MM, explore their mechanisms of action, and describe the develop- ment of a predictive assay for PIKfyve sensitivity.
Methods
PIKfyve inhibitor sensitivity
APY0201 was included in a 76-drug panel high throughput screen and evaluated in a 7-point, 10-fold dilution of drug con- centration, starting at 10 μM. Twenty-five human MM cell lines (HMCL) and 15 NHL cell lines were incubated for 24 or 72 h. Cellular viability was assessed with the CellTiter Glo (Promega) assay for all dose-response curves. Mid-point half maximal effective concentrations (herein denominated EC50), maximum inhibition, and area under the curve (AUC) were calculated.17
Twenty HMCL were treated with a 20-point 2-fold dilution of drug concentration, starting at 40 μM, and incubated for 72 h with APY0201 (MedChemExpress, HY-15982, Monmouth Junction, NJ, USA), apilimod (Santa Cruz Biotechnology, sc-480051, Dallas, TX, USA), and YM201636 (SelleckChem, S1219, Houston, TX, USA).
Ex vivo sensitivity to APY0201 was assessed after 24 h incu- bation in 100 purified patient-derived MM samples (through magnetic bead sorting for CD138+ cells; average purity greater than 95%). Fifteen samples were also screened against APY0201 and apilimod in a 14-point, 3-fold dilution of drug concentration, starting at 50 μM, and incubated for 72 h. Leukocytes from whole bone marrow samples were incubated for 24 h with increasing concentrations of APY0201 to measure cytotoxicity, as described previously.18 Written informed con- sent was obtained from the patients and samples were collected and stored under Mayo Clinic Institutional Review Board approval (IRB 919-04, 2207-02, 15-009436, and 18-003198). This study was conducted in accordance with the Declaration of Helsinki.
Immunoblotting
Anti-β-actin (#A00702-100) antibody was purchased from GeneScript (Piscataway, NJ, USA), anti-Lamp-1 (#ab25630) was purchased from Abcam (Cambridge, MA, USA), anti-SQSTM1 (#sc-28359) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA), and anti-cathepsin A (#AF1049) and anti- cathepsin D (#AF1014) were purchased from R&D Systems (Minneapolis, MN, USA). Antibodies against β-tubulin (#2128), Beclin1 (#3495), Caspase 3 (#9662), GAPDH (#2118), Lamin A/C (#4777), LC3A/B (#12741), PARP (#9542), and transcription factor EB (TFEB, #4240) were purchased from Cell Signaling Technology (Danvers, MA, USA).
Autophagy organelle formation
Vacuolar phenotype was evaluated by live cell differential interference contrast (DIC) imaging. Acidic vacuoles were identified with the LysoSensor Yellow/Blue DND-160 probe (#L7545, Thermo Fisher Scientific, Waltham, MA, USA). HMCL were incubated with 20 μL of the Premo Autophagy Tandem Sensor RFP-GFP-LC3 kit (#P36239, Thermo Fisher Scientific) for 48 h with subsequent addition of dimethylsul- foxide (DMSO) control or APY0201 for 18 h. The Autophagy Detection Kit (Abcam #ab139484) was used according to the manufacturers’ recommendations. Cellular viability was meas- ured at 72 h.
mRNA sequencing
HMCL were treated with 100 nM and patients’ sam- ples with 50 and 500 nM of APY0201, and DMSO as a control, for 24 h; the cells were then harvested, and total RNA was extracted. An internally developed RNA- sequencing analysis workflow (MAPRSeq,19 v3.0.1) was used to perform comprehensive analysis of raw RNA sequencing paired-end reads, which were aligned by a fast and splice-aware aligner (STAR,20 v 2.5.2b) to human genome build hg38. Quality control was performed with RSeQC (v3.0.0).21 Gene expression was quantified with featureCounts22 from the Subread package (http://sub- read.sourceforge.net/, v1.5.1). Genes were determined as differentially expressed based on log-fold change >1, and the Enrichr platform23 was used to identify involved pathways. The data have been deposited in the National Center for Biotechnology Information’s (NCBI) Gene Expression Omnibus (GEO),24 accessible through GEO series accession number GSE134598.
Results
Identification of PIKfyve as a potential target
in multiple myeloma and non-Hodgkin lymphoma
Medium throughput screening on MM and NHL cell lines was first conducted at Nanosyn Inc. (Santa Clara, CA, USA) using a panel of known Food and drug Administration-approved oncology drugs, kinase inhibitors, and epigenetic compounds assembled by the investigators. A more refined panel of 76 drugs was then selected and sensitivity to the single agents was evaluat- ed in 25 MM and 15 NHL cell lines. We observed a dose dependent inhibition of cellular viability in all of the 25 HMCL exposed to the PIKfyve inhibitor APY0201 after 72 h incubation, with a median EC50 of 55 nM and a median maximum inhibition of cellular viability of 81% (dose-response curves are shown in Online Supplementary Figure S1). These results prompted a confirmatory valida- tion screen of 20 HMCL treated with APY0201 and two additional PIKfyve inhibitors, YM201636 and apilimod, with an increased dose range. Dose-dependent inhibition of cellular viability was observed in this confirmatory screen in 20 HMCL with all three PIKfyve inhibitors after 72 h incubation. APY0201 was the most potent PIKfyve inhibitor, followed by YM201636 and apilimod (Table 1, Online Supplementary Figures S2-S4 and Online Supplementary Table S1).
In NHL cell lines the effect was similar; after 72 h incu- bation, APY0201 was the most potent PIKfyve inhibitor evaluated, with 14 of 15 tested lines (93%) having an EC50 in the nanomolar range (median 76 nM). For YM201636, only 37.5% of eight tested NHL cell lines were sensitive at nanomolar concentrations with a medi- an EC50 of 530 nM. Finally, for apilimod, only one of eight tested NHL lines (12.5%) had nanomolar activity with an EC50 of 405 nM (Online Supplementary Figures S5 and S6 and Online Supplementary Table S2).
Ex vivo anti-myeloma activity of multiple PIKfyve inhibitors
We next tested 100 ex vivo primary CD138+-selected patients’ samples. Dose-dependent sensitivities for APY0201 were observed in 40% of these ex vivo samples at 24 h, with 47% of the responsive samples exhibiting an EC50 lower than 100 nM (19% of all tested samples). The
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