CXCR4 Loss Reduces the Local Growth of Richter Syndrome Murine and Patient-Derived Xenograft Models and Impairs BCR and VLA-4 Activation

Abstract

Richter Syndrome (RS) represents transformation of chronic lymphocytic leukemia (CLL) into an aggressive lymphoma and is considered the most important unmet clinical need in CLL because of the lack of effective treatments. An important feature of RS cells is the frequent acquisition of genetic lesions in cell cycle regulators, such as CDKN2A/CDKN2B and TP53, resulting in increased proliferative capacity of the transformed cells and reduced dependence on proliferative microenvironmental signals. However, interactions with the tumor microenvironment are still required to promote the expansion of the transformed cells, as evidenced by the delayed growth of murine TCL1-derived or patient-derived xenograft (PDX) RS models following BCR disruption or macrophage depletion (Chakraborty S et al. Blood 2021, Martines C et al. Blood 2022). To further define the relevance of individual microenvironmental signals in supporting the growth of RS cells in vivo, in this study we evaluated the effects of CRISPR/Cas9-mediated knockout of the chemokine receptor CXCR4, which plays a central role in regulating the homing and interaction with the tumor microenvironment of non-transformed CLL cells. CRISPR/Cas9 editing of CXCR4 was done in the murine TCL1-derived RS model TC1-355 TKO and in the RS-PDX model RS9737, which are both characterized by biallelic disruption of TP53, CDKN2A and CDKN2B. The edited cells, comprising a mixed population with more than 80% disrupted CXCR4 alleles, were then transplanted in the peritoneal cavity and subcutaneous tissue of recipient mice (n=3 C57BL/6 mice for TCL1-355 TKO, n=8 NSG mice for RS9737). Analysis of the mutant allele frequency several weeks after injection revealed a significant reduction in the proportion of cells with mutated CXCR4 in the spleens of the injected mice and to a lesser extent in the peritoneal cavity and subcutaneous tumors, suggesting that disruption of CXCR4 signaling affects not only the migration and homing of the malignant B cells but also their local growth. To further evaluate the latter possibility, we transplanted CXCR4 knockout and CXCR4 wild type RS9737 cells in separate flanks of NSG mice (n=8) and after 4 weeks investigated the percentage of proliferating cells in the tumors by in vivo BrdU incorporation assay. A significantly lower percentage of proliferating cells was observed in the CXCR4 knockout compared to CXCR4 wild type tumors (22.3 ± 2.1 vs 37 ± 1.4, respectively, p<0.001), further suggesting that loss of CXCR4 affects the proliferation of the tumor cells. Since CXCL12 stimulation had no effect on the in vitro proliferation rate of the RS cells, we next investigated whether the reduced in vivo proliferation could be an indirect effect. For this purpose, we investigated whether CXCR4 knockout would affect integrin VLA4 or BCR signaling, as both BCR and CXCR4 can mediate inside-out VLA4 activation and there are data suggesting a potential crosstalk between the BCR and CXCR4 pathways (Maity PC et al. Front Immunol. 2018). Analysis of BCR signaling in the TCL1-355 TKO model showed a significant reduction in anti-IgM induced activation of AKT, ERK and reduced calcium mobilization in the CXCR4 knockout cells despite increased surface IgM expression. However, no effect of CXCR4 knockout on BCR signaling was observed in RS9737 cells, whereas CXCL12-induced VLA4 activation and VCAM1 adhesion were impaired in both models by CXCR4 knockout. To investigate whether impaired VLA4 activation could be responsible for the reduced in vivo proliferation rate of CXCR4 knockout RS cells, we performed BrdU incorporation analysis of RS9737 CXCR4 knockout and CXCR4 wild type cells co-cultured with the human stromal cell line HS5 + CXCL12. Co-culture with HS5 + CXCL12 resulted in a significantly increased proliferation of RS9737 CXCR4 wild type cells compared to the unstimulated control condition (58.5% ± 0.9% vs 50.1% ± 4.3%, respectively, p=0.031), whereas no such difference was observed with RS9737 CXCR4 knockout cells (50.0% ± 1.5% vs 49.1% ± 0.5%, respectively, p=n.s.). Together, these data show that CXCR4 disruption results in negative selection and reduced growth of murine and PDX RS models in vivo and affects BCR and VLA-4 signaling, suggesting that the CXCR4 pathway could represent a therapeutic vulnerability in Richter Syndrome.