NSD2 overexpression increases MM cell dependence on AK2. (A) Plot of MAGeCK-generated AK2 Beta scores and the corresponding FDRs in NTKO and TKO cells. (B) Depmap AK2 dependency scores across different cancer cell line lineages (red - MM cell lines; blue - other hematologic cell lines; grey - solid cancer cell lines). t(4;14) MM cell lines are labeled. (C) Comparison of AK2 dependency scores between t(4;14) and non-t(4;14) MM cell lines. In B and C, horizontal lines represent median gene effect scores. ∗P < .05 Mann-Whitney, one-sided. (D) Kaplan-Meier curves showing overall survival (OS and PFS of patients with MM stratified by AK2 expression into low (Q1; blue), mid (Q2+Q3; green), and high (Q4; red). P value were calculated by Cox proportional hazards regression. (E) Hazard ratio of AK2 expression associated with OS and PFS in t(4;14) (red) and other (blue) patients with MM as determined using Cox proportional hazards regression. Lines represent 95% confidence intervals RNAseq and WGS. P value and cohort sizes are shown (total N = 725, baseline patients with CoMMpass). (F) Schematic representation of confirmatory in vitro competitive growth assays used for validating essential genes. (G) Time-dependent depletion of GFP-positive NTKO and TKO isogenic cells after transduction with LentiCRISPRv2-GFP expressing the indicated sgRNAs. sgROSA and sgPCNA were used as negative and positive controls, respectively. (H) Comparison of time-dependent depletion of GFP-positive cells harboring sgAK2 in NTKO, TKO, and NSD2-replete TKO cells. (I) Competitive growth of nonlabeled NTKO and GFP-labeled TKO cells treated with DMSO or the AK2 inhibitor AP5A (50 μM) for 72 h. (J) Time-dependent depletion of control and NSD2-depleted KMS34 cells after transduction with LentiCRISPRv2-GFP expressing the indicated sgRNAs. Cells were transduced with LentiCRISPRv2 expressing a nontargeting (sgNT) or NSD2-targeting (sgNSD2) sgRNA and selected with puromycin before transduction with the LentiCRISPRv2-GFP constructs. All experiments were performed in biological triplicate. (K) Time-dependent depletion of t(4;14) and non-t(4;14) MM cell lines transduced with inducible Cas9/GFP/sgAK2 (TLCV2) vectors. Percentage of GFP-positive cells were measured at the indicated times by flow cytometry. Experiments were performed in biological duplicate. (L) Subcutaneous flank injections of cells expressing doxycycline-inducible scrambled (shSc) or AK2-targeting (shAK2) shRNA into NOD/SCID mice. Mice were fed a doxycycline chow diet. (M) KMS11-derived tumors isolated from the left (shSc) and right (shAK2) mouse flanks. Tumor dimensions were measured using a caliper and the volumes were calculated using the following formula: V = ½ L x W2. (N) Tumors derived from shSc-expressing LP1 cells. No tumors from AK2-depleted LP1 cells were detected. Immunoblots confirming continued AK2 expression in isolated LP1-derived tumors are shown. ∗∗P < . 01, ∗∗∗P < . 001, and ∗∗∗∗P < . 0001. DMSO, dimethyl sulfoxide; sgROSA, sgRNA targeting mouse Rosa26 locus; sgPCNA; TLCV2, LentiCRISPRv2 with Tet Response Element Promoter; WGS, whole genome sequencing.
