A calcium- and calpain-dependent pathway determines the response to lenalidomide in myelodysplastic syndromes. Homo sapiens

Despite the high response rates of individuals with myelodysplastic syndrome (MDS) with deletion of chromosome 5q (del(5q)) to treatment with lenalidomide (LEN) and the recent identification of cereblon (CRBN) as the molecular target of LEN, the cellular mechanism by which LEN eliminates MDS clones remains elusive. Here we performed an RNA interference screen to delineate gene regulatory networks that mediate LEN responsiveness in an MDS cell line, MDSL. We identified GPR68, which encodes a G-protein-coupled receptor that has been implicated in calcium metabolism, as the top candidate gene for modulating sensitivity to LEN. LEN induced GPR68 expression via IKAROS family zinc finger 1 (IKZF1), resulting in increased cytosolic calcium levels and activation of a calcium-dependent calpain, CAPN1, which were requisite steps for induction of apoptosis in MDS cells and in acute myeloid leukemia (AML) cells. In contrast, deletion of GPR68 or inhibition of calcium and calpain activation suppressed LEN-induced cytotoxicity. Moreover, expression of calpastatin (CAST), an endogenous CAPN1 inhibitor that is encoded by a gene (CAST) deleted in del(5q) MDS, correlated with LEN responsiveness in patients with del(5q) MDS. Depletion of CAST restored responsiveness of LEN-resistant non-del(5q) MDS cells and AML cells, providing an explanation for the superior responses of patients with del(5q) MDS to LEN treatment. Our study describes a cellular mechanism by which LEN, acting through CRBN and IKZF1, has cytotoxic effects in MDS and AML that depend on a calcium- and calpain-dependent pathway. Overall design: Loss of function screen was performed with GeneNetTM Lentiviral shRNA Libraries (System Biosciences, S12XXB-1) and analyzed by GeneNet Library Data Analysis Software (System Biosciences) according to the manufacturer. Briefly, VSV-G pseudotyped lentiviral particles targeting ~47,000 transcripts in the human genome according to the NCBI RefSeq database (GRCh38.p6) were transduced into 2 x 10^6 MDSL cells with MOI at 0.5–1. Within this range, each cell that has been successfully transduced is expected to contain only one copy of a given shRNA construct. Three days post transduction, GFP+ transduced MDSL cells were purified for drug selection with DMSO or 10 μM LEN for seven days. 5 x 10^6 viable MDSL cells were then isolated for RNA extraction. After two rounds of PCR, the amplified cDNA corresponding to the unique shRNA hairpins were labeled with biotin. After removing the sense strands, the biotin-labeled antisense strands were used as hybridization targets for Human Genome U133+2.0 GeneChip® Arrays (Affymetrix, #900470). GeneNet Library Data Analysis Software (System Biosciences) was used to generate expression values for individual shRNAs by comparing the background corrected and normalized signal values for each shRNA in LEN treated and control samples. The list of enriched shRNAs was calculated based on the absolute mean difference of expression values of shRNAs in LEN versus control treated replicate samples. Only shRNAs with > 1.5-fold enrichment were further considered. P values were calculated based on the biological replicates of individual shRNA clones using Student’s t-test. To further narrow the list of hits for adjusted P values, False Discovery Rate (FDR) was applied.