Abstract Deletion of the RB1 gene, which encodes a tumor suppressor that inhibits cell cycle progression, is found in approximately 15% of chronic lymphocytic leukemia (CLL) patients and represents one of the most frequent genetic lesions in this disease. RB1 deletions typically occur in the context of large 13q14 deletions, which invariably involve the minimal deleted region (MDR) encompassing the miR-15a/16-1 and DLEU2 genes. Functional evidence for a causative role of the MDR deletion in CLL pathogenesis has been provided by knockout mouse models, but similar functional studies targeting RB1 are lacking. In addition, no data are available regarding the potential role of RB1 deletions in the pathogenesis of Richter's Transformation (RT), which is characterized by frequent genetic lesions that result in cell cycle deregulation. Most notably, deletions of the cyclin-dependent kinase inhibitors CDKN2A/2B are seen in up to 50% of RT tumors, typically in combination with TP53 abnormalities. In a recent study, we showed that concurrent loss of CDKN2A/2B and TP53 in normal murine B cells results in the development of CLL-like B cell lymphoproliferations, with histological features of RT present in the majority of cases (Martines C et al, Blood, 2024, 144:759). Given the related role of RB1 in cell cycle regulation, its loss would be expected to have similar functional consequences. To further explore this possibility, we used multiplex CRISPR/Cas9 editing to introduce loss-of-function mutations in RB1 and TP53 into splenic B cells isolated from healthy C57BL/6 mice or mice carrying the 13q14-MDR deletion. Edited cells were transplanted into NSG mice (n=14 for C57BL/6-derived B cells; n=10 for 13q14-MDR-derived B cells), which were monitored for disease development by peripheral blood flow cytometry analysis. After a median follow-up of 132 days, 12/24 (50%) recipient mice developed a B cell leukemia. Disease onset occurred significantly earlier and more frequently in mice receiving 13q14-MDR-derived B cells (n = 8/10; median 126 days) compared to C57BL/6-derived B cells (n = 4/14; median 151 days; p = 0.0028), suggesting functional synergy between RB1 and TP53 loss and 13q14-MDR deletion. Flow cytometry analysis revealed that in 8 cases, the leukemia cells displayed a CLL-like immunophenotype (surface expression of CD5, CD19, CD20, CD22, CD38 and IgM), while the remaining 4 leukemias showed a CD5-negative mature B cell immunophenotype (CD19+CD20+CD22+CD38+IgM+). Histological evaluation of spleen samples from 9 recipient mice showed an infiltrate of small-to-medium sized lymphocytes in 5 cases (4 CD5-positive and 1 CD5-negative), while the other 4 were characterized by sheets of large, diffusely infiltrating cells, consistent with an RT-like histological pattern (observed in 2 CD5-positive and 2 CD5-negative tumors). The above findings demonstrate that combined loss of RB1 and TP53 can result in the development of both CLL and RT-like disease, as previously observed for the CDKN2A/2B/TP53 combination. However, comparing the time to disease onset with our prior CDKN2A/2B/TP53 knockout model (median 105 days), the RB1/TP53 malignancies exhibited slower kinetics (median 151 days; p = 0.055). To further investigate the differential impact of CDKN2A/2B vs. RB1 loss in CLL, these and other common CLL drivers were investigated via multiplex ligation-dependent probe amplification in a series of 377 CLL patients followed at a single institution. Overall, 9 (2.4%) patients had deletion of CDKN2A/2B and 51 (13.5%) had RB1 deletion, most of which in the context of a large del13q14 (n=49). Follow-up clinical data were available for all 9 patients with CDKN2A/2B and 38 patients with RB1 deletion. Survival analysis showed a significantly shorter overall survival (OS) for patients with CDKN2A/2B deletion compared to patients with RB1 deletion, both when calculated from diagnosis (median OS 74 months vs 137 months, respectively, p=0.01) and from the detection of the genetic lesion (median OS 53 months vs 65 months, respectively, p=0.017). Collectively, the above data provide further evidence that RB1 deletion represents an important CLL driver but reveal differences in disease kinetics and outcomes with respect to the functionally related CDKN2A/2B deletion. The underlying differences, as well as mechanisms determining the commitment towards a particular phenotype (i.e., CLL- vs. RT histology) are currently being investigated.
Jauk et al. (Mon,) studied this question.
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