Loss of Function of The Cell Cycle Regulator Cdh1 Causes Cell Fragility due to Aberrant G2/M Checkpoint and Develops Resistant Disease in a B-ALL/LBL Mouse Model
Authors: Ishizawa Jo , Sugihara Eiji , Hashimoto Norisato , Kuninaka Shinji , Kojima Kensuke , Andreeff Michael , Okamoto Shinichiro , Saya Hideyuki 
Published: October 2019

Abstract
Cdh1, one of the co-activators for anaphase-promoting complex/cyclosome, plays a crucial role in the mitotic phase, but also has been reported as G2/M checkpoint regulator activated by irradiation-induced DNA damage. Focusing on Cdh1 functions in the hematopoietic system, we have generated Cdh1 conditional gene-trap (Cdh1f/f) mice and crossed them with Mx1-Cre transgenic mice to obtain Mx1-Cre (+) / Cdh1f/f mice. These animals illustrate that the irradiation-induced G2/M checkpoint is defective in Cdh1-deficient bone marrow (BM) cells, which causes the loss of stem/progenitor cells through mitotic catastrophe (Jo Ishizawa et al, Cancer Science 2011). We have also generated a Cdh1-deficient B cell lymphoblastic leukemia/lymphoma (B-ALL/LBL) mouse model, and reported that it is a promising model to investigate if Cdh1 affects the prognosis and therapy sensitivity of B-ALL/LBL.
We here report, with our mouse model, that Cdh1 downregulation causes cell fragility in lymphoma cells possibly due to aberrant G2/M checkpoint, which ultimately causes the generation of more resistant phenotype yeilding a poorer disease prognosis after secondary and tertiary transplantation.
We transduced Myc oncogene into Cdh1-intact and Cdh1-deficient BM mononuclear cells (BM-MNCs) and transplanted Myc-transduced BM-MNCs (GFP+) into sub-lethally irradiated wild type C57BL/6 mice, which developed B-ALL/LBL phenotype with similar incidence rates (70% versus 80%; p = 0.72, n = 20). There were no differences in leukemia cell morphology and immunophenotype between Cdh1-intact and Cdh1-deficient mice (CD3-/B220+/IgM-/Mac1-/Gr1- uniform blast cells with > 95% Ki67+). These results showed that Cdh1 is dispensable for Myc-related lymphoid leukemogenesis.
The survival of Cdh1-deficient B-ALL/LBL mice was slightly, but statistically significantly longer than the Cdh1-intact mice (median survival: 78 days versus 95 days in the Cdh1-deficient group; p = 0.0064, n = 14 and 9, respectively). In addition, histologic sections of affected lymph nodes showed more prominent “starry sky” pattern (focal cell death area) in the Cdh1-deficient group (Fig. 1, %area; 1.126 ± 0.083 versus 3.353 ± 0.788, p < 0.05, n = 3 for each), indicating that the cell fragility probably causes the better prognosis in this group. Furthermore, TUNEL-negative dead cells were observed in the Cdh1-deficient mice, suggesting non-apoptotic cell death.

We conducted DNA microarray analysis using GFP+ tumor cells derived from the bone marrow of Cdh1-intact or -deficient B-ALL/LBL mice (n = 3 for each). Biotin-labeled aRNA probes were synthesized from the total RNA and subjected to hybridization with a Mouse Genome 430 2.0 Array, Affymetrix. In GSEA analysis, three gene sets related to mitotic phase and DNA damage response were listed up in high normalized enrichment score (NES; 1.719 in “G2/M checkpoints”, 1.603 in “activation of ATR in response to replication stress” and 1.593 in “mitotic prometaphase”, p < 0.05). Furthermore, pHH3 or γH2A.X staining in the lesions showed that the event numbers of pHH3 or γH2A.X-positive cells in cell death area were significantly increased in Cdh1-deficient lymph node lesions (n=3, 3.0±1.0 vs 23.3±7.02/mm3, p < 0.01), indicating that the Cdh1-dificient tumor cells could be easily killed during mitotic phase due to DNA damage.
In order to investigate if Cdh1 loss affects the maintenance of B-ALL/LBL, we conducted secondary and tertiary transplantation. The prognosis apparently reversed, that is, the Cdh1-deficient group showed poorer prognosis in both of secondary and tertiary transplants (Fig. 2A, n = 6 for each, p = 0.0248 and p = 0.0004, respectively). Furthermore, we irradiated (4Gy) the mice on day 14 post-secondary transplantation and found that the Cdh1-deficient B-ALL/LBL mice were more radio-resistant than the others (Fig. 2B). These results can explain the hypothesis that Cdh1 loss-induced genetic instability due to abnormal G2/M checkpoint generated clones that were more resistant and had enhanced disease progression.

In conclusion, Cdh1 plays a crucial role in protecting Myc-induced B-ALL/LBL cells from genotoxic stress-induced cell death, probably as G2/M checkpoint regulator, and Cdh1 loss causes the generation of more resistant clones due to genetic instability.

Disclosures:
No relevant conflicts of interest to declare.