Figure 7.
CDC20 expression is reduced in CLL and correlates with a more aggressive cell and disease phenotype. (A) CDC20 expression in different lymphatic and myeloid neoplasms as measured by microarray-based gene expression profiling (data set of Haferlach et al).50 Boxes with medians and 25th to 75th percentiles. Whiskers show 10th and 90th percentiles. CLL stands out among the other hematopoietic cancers, as its CDC20 expression is markedly reduced; for example, FCh = −6.8; P < .001 compared with healthy controls. (B) Expression of TCL1A and CDC20 in primary human CLL cells. Representative immunoblot showing decreased CDC20 expression in lysates from unstimulated U-CLL (N = 6) vs M-CLL (N = 6) (left). Linear regression analysis demonstrates an inverse correlation of TCL1A and CDC20 protein levels in CLL (N = 19; P = .002; R = 0.437) (right). (C) GEP of previously untreated patients with CLL (N = 337) of the prospective CLL8 trial identified an inverse correlation of TCL1A (blue) and CDC20 (green) expression. (D) Patients from the CLL8 trial were divided into 2 groups by the median expression of CDC20. Patients with low CDC20 expression (N = 166, red) showed significantly higher WBC counts in comparison with those with high CDC20 (N = 163, blue). Boxes show medians and the 25th to 75th percentiles, whiskers the 10th to 90th percentiles. Significance was determined using the median test. (E) Patients from the CLL8 trial that received fludarabine/cyclophosphamide (FC) were divided into 2 groups by the median expression of CDC20. The Kaplan-Meier curve illustrates the significantly shorter progression-free survival (PFS) of patients with low CDC20 expression (N = 86; green; median PFS, 25.5 months) compared with those with high CDC20 expression (N = 83; blue; median PFS, 34.1 months); P = .014, log-rank test. (F) CDC20 expression levels are significantly lower in CLL that we previously characterized by a molecular profile of genome instability (N = 189)29 than in those with activation of epithelial-mesenchymal transition (EMT)–like programs (N = 130; P < .001, Mann-Whitney test). Boxes with medians, 25th to 75th percentiles, and whiskers of mininum/maximum. (G) Reanalysis of single-cell RNA sequencing data from Eμ-TCL1A and Eμ-TCL1AAkt-C (Richter syndrome model) mice that we published in Kohlhaas et al.30 The UMAP displays the clusters identified in the integrative analysis of both models that were then applied to the Eμ-TCL1A model only (N = 4 mice). Clusters 2 and 10 are enriched in Eμ-TCL1A cells, whereas clusters 0, 3, 13, 18, 19, and 20 represent Eμ-TCL1AAkt-C or those that are shared with Eμ-TCL1A. (H) Cdc20 is primarily expressed in the Eμ-TCL1A enriched Seurat cluster 2. (I) TCL1A expression is significantly lower in Seurat cluster 2 compared with all other clusters, P < .001 (supplemental Table 16). (J) Setup of in vivo Cdc20 knockdown experiment: leukemic Eμ-TCL1A splenocytes51 were nucleofected with the transposon-based pJ547-shCdc20 construct. It encodes a GFP cassette with microRNA 30 (miR-30)–based shRNA sequences against murine Cdc20 at the 3′ end. A pJ547-GFP control encoded an unspecific shRNA sequence. After intraperitoneal injection into syngeneic hosts and tumor development, GFP+ splenocytes were purified using fluorescence-activated cell sorter (FACS) and reinjected into hosts. Knockdown efficiency was tested by immunoblots (right). (K) Flow-cytometric analysis of PB cells showing a faster increase of the aberrant leukemic Cd5+ Cd19+ B-cell population in recipients that were transplanted with Eμ-TCL1A;Cdc20-KD B cells (N = 19) compared with Eμ-TCL1A;GFP control animals (N = 15). Boxes indicate medians and 25th to 75th percentiles and whiskers show minimum and maximum; significance was tested using a two-way analysis of variance with Bonferroni correction for multiple testing. (L) Immunoblot analysis showing increased cyclin D1 and slightly reduced PARP cleavage in splenocytes from Eμ-TCL1A;Cdc20-KD compared with Eμ-TCL1A;GFP mice. Splenocytes were isolated at the end point of survival analysis. Each lane represents an individual animal. (M) The proportion of metaphases with an aberrant number of chromosomes was significantly higher in Eμ-TCL1A;Cdc20-KD cells compared with Eμ-TCL1A;GFP cells (Student t test). AML, acute myeloid leukemia; B-ALL, B-cell acute lymphoblastic leukemia; CML, chronic myeloid leukemia; hygro, hygromycin resistance; MDS, myelodysplastic syndrome; ped., pediatric.

CDC20 expression is reduced in CLL and correlates with a more aggressive cell and disease phenotype. (A) CDC20 expression in different lymphatic and myeloid neoplasms as measured by microarray-based gene expression profiling (data set of Haferlach et al).50 Boxes with medians and 25th to 75th percentiles. Whiskers show 10th and 90th percentiles. CLL stands out among the other hematopoietic cancers, as its CDC20 expression is markedly reduced; for example, FCh = −6.8; P < .001 compared with healthy controls. (B) Expression of TCL1A and CDC20 in primary human CLL cells. Representative immunoblot showing decreased CDC20 expression in lysates from unstimulated U-CLL (N = 6) vs M-CLL (N = 6) (left). Linear regression analysis demonstrates an inverse correlation of TCL1A and CDC20 protein levels in CLL (N = 19; P = .002; R = 0.437) (right). (C) GEP of previously untreated patients with CLL (N = 337) of the prospective CLL8 trial identified an inverse correlation of TCL1A (blue) and CDC20 (green) expression. (D) Patients from the CLL8 trial were divided into 2 groups by the median expression of CDC20. Patients with low CDC20 expression (N = 166, red) showed significantly higher WBC counts in comparison with those with high CDC20 (N = 163, blue). Boxes show medians and the 25th to 75th percentiles, whiskers the 10th to 90th percentiles. Significance was determined using the median test. (E) Patients from the CLL8 trial that received fludarabine/cyclophosphamide (FC) were divided into 2 groups by the median expression of CDC20. The Kaplan-Meier curve illustrates the significantly shorter progression-free survival (PFS) of patients with low CDC20 expression (N = 86; green; median PFS, 25.5 months) compared with those with high CDC20 expression (N = 83; blue; median PFS, 34.1 months); P = .014, log-rank test. (F) CDC20 expression levels are significantly lower in CLL that we previously characterized by a molecular profile of genome instability (N = 189)29 than in those with activation of epithelial-mesenchymal transition (EMT)–like programs (N = 130; P < .001, Mann-Whitney test). Boxes with medians, 25th to 75th percentiles, and whiskers of mininum/maximum. (G) Reanalysis of single-cell RNA sequencing data from Eμ-TCL1A and Eμ-TCL1AAkt-C (Richter syndrome model) mice that we published in Kohlhaas et al.30 The UMAP displays the clusters identified in the integrative analysis of both models that were then applied to the Eμ-TCL1A model only (N = 4 mice). Clusters 2 and 10 are enriched in Eμ-TCL1A cells, whereas clusters 0, 3, 13, 18, 19, and 20 represent Eμ-TCL1AAkt-C or those that are shared with Eμ-TCL1A. (H) Cdc20 is primarily expressed in the Eμ-TCL1A enriched Seurat cluster 2. (I) TCL1A expression is significantly lower in Seurat cluster 2 compared with all other clusters, P < .001 (supplemental Table 16). (J) Setup of in vivo Cdc20 knockdown experiment: leukemic Eμ-TCL1A splenocytes51 were nucleofected with the transposon-based pJ547-shCdc20 construct. It encodes a GFP cassette with microRNA 30 (miR-30)–based shRNA sequences against murine Cdc20 at the 3′ end. A pJ547-GFP control encoded an unspecific shRNA sequence. After intraperitoneal injection into syngeneic hosts and tumor development, GFP+ splenocytes were purified using fluorescence-activated cell sorter (FACS) and reinjected into hosts. Knockdown efficiency was tested by immunoblots (right). (K) Flow-cytometric analysis of PB cells showing a faster increase of the aberrant leukemic Cd5+ Cd19+ B-cell population in recipients that were transplanted with Eμ-TCL1A;Cdc20-KD B cells (N = 19) compared with Eμ-TCL1A;GFP control animals (N = 15). Boxes indicate medians and 25th to 75th percentiles and whiskers show minimum and maximum; significance was tested using a two-way analysis of variance with Bonferroni correction for multiple testing. (L) Immunoblot analysis showing increased cyclin D1 and slightly reduced PARP cleavage in splenocytes from Eμ-TCL1A;Cdc20-KD compared with Eμ-TCL1A;GFP mice. Splenocytes were isolated at the end point of survival analysis. Each lane represents an individual animal. (M) The proportion of metaphases with an aberrant number of chromosomes was significantly higher in Eμ-TCL1A;Cdc20-KD cells compared with Eμ-TCL1A;GFP cells (Student t test). AML, acute myeloid leukemia; B-ALL, B-cell acute lymphoblastic leukemia; CML, chronic myeloid leukemia; hygro, hygromycin resistance; MDS, myelodysplastic syndrome; ped., pediatric.

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