Fig 2: CKS1B and CKS2 expression is higher in diagnostic bone marrow samples from patients carrying MLL-translocations compared to PBMCs from healthy controls. RNA extracted from human umbilical cord blood CD34+ cells, PBMCs from healthy donors, and leukaemic patient samples, were assessed for (A) CKS1B and (B) CKS2 expression by TaqMan quantitative PCR. Three independent control genes were used (ABL, GUS, B2M) to measure relative RNA abundance. Values are represented as relative gene expression versus GUS control. Each point represents one patient. Median bars with standard deviation are presented for each set of samples. A Student's t-test was used to assess significance, and * indicates significance versus CD34+ cells, and § indicates significance versus PBMCs.

Fig 3: MLL-translocation cell lines are sensitive to inhibition of pan-Cullin-dependent and CKS1-dependent protein degradation. (A) Western blots for THP-1 (MLL-AF9) and KOPN-8 (MLL-ENL) cells treated with vehicle control (DMSO), 0.1 µM MLN4924 or 1 µM C1 for 16 h and 24 h. Histone H3 was used as a loading control. Western blots are representative of 3 independent experiments. (B) Cell viability was assessed for THP-1, KOPN-8, healthy PBMC and CD34+ control cells after 3 days treatment with indicated concentrations of the pan-Cullin inhibitor (MLN4924) or the SKP2-CKS1 inhibitor (C1). Results represent the mean of 3 independent experiments with standard deviation bars. (C) Proportion of apoptotic CD34+, THP-1 and KOPN-8 cells, in response to two different concentrations of C1 or MLN4924, was measured by PI and Annexin V-FITC staining using flow cytometry. Significance was tested using the Student's t-test versus DMSO treated cells (N = 3). (D) Cell cycle status was measured for KOPN-8 cells in response to DMSO vehicle control or MLN4924 (1 µM) using the Click-iT EdU incorporation kit. Graphs are representative of 3 independent experiments.

Fig 4: Modulation of Wnt signalling in MLL-translocation cell lines by CKS1 and CKS2. TCF/LEF activity was measured in (A) KOPN-8 (MLL-ENL) and (B) THP-1 (MLL-AF9) cell lines using the TOPFlash reporter plasmid (or FOPFlash plasmid control) and pRL-TK Renilla control. Cell lines were co-transfected with siRNA to knockdown expression of CKS1B, CKS2 or CKS1B + CKS2. Values are represented as fold change RLA versus Non-specific siRNA treated cells. Significance was tested using the Student's t-test (N = 3).