The Expression of the GTPase-Deficient, Hematopoietic-Specific RhoH GTPase Is Implicated in Development of Chronic Lymphocytic Leukemia (CLL).

Abstract

RhoH is a hematopoietic-specific small GTPase which is constitutively active due to lack of intrinsic GTPase activity, suggesting that expression levels of RhoH regulate cellular function. Our lab has previously shown that RhoH in T-cells directly interacts with ZAP-70, a critical protein tyrosine kinase in TCR cell signaling and function (Gu et al., Nature Immunol., 2006). As in human B-CLL ZAP-70 expression has been correlated with an unmutated IgVH gene status and poor prognosis, we have investigated RhoH expression and function in B-CLL. PBMC samples from 29 B-CLL patients, all containing >90% CD19+/CD5+ human B-CLL cells, were compared to CD19+ B-cells from normal donors. Significantly higher RhoH mRNA levels were detected in CLL samples by quantitative PCR (p<0.001) with a 2–4 fold increase in the majority of samples. In addition, RhoH levels in these CLL samples were directly correlated with ZAP-70 expression (p<0.01). To further delineate the role of RhoH in the development and progression of B-CLL, we utilized a genetic mouse model of B-CLL, the Eμ-TCL1 transgenic mouse (Bichi et al. PNAS, 2002), and interbred these with Rhoh−/− mice. Starting at 2 months of age peripheral blood of these mice was analyzed every month for different blood cell lineages and in particular for the number of IGM+/CD5+/B220low cells as a readout for the progression of the CLL-like disease. In TCL1 transgenic mice with normal RhoH expression (Rhohwt; TCL1Tg) and with one Rhoh allele knocked out (Rhoh+/−; TCL1Tg) an accumulation of IgM+/CD5+ B-cells was observed which started as early as an age of 3 and 4 months, respectively, and increased dramatically thereafter. In contrast, Rhoh−/−; TCL1Tg showed no significant increase of IgM+/CD5+ B-cells until month 5 with a modest increase thereafter and values were significantly reduced in comparison to Rhohwt; TCL1Tg from month 3–5. As expected, no increase of IgM+/CD5+ B-cells was observed in the nontransgenic controls Rhoh−/+; TCL1NTg, Rhoh−/−; TCL1NTg (data not shown), and Rhohwt; TCL1NTg (Table 1).Table 1:IGM+/CD5+ cells in the peripheral blood.Age [months]Rhohwt; TCL1TgRhoh+/−;TCL1TgRhoh−/−;TCL1TgRhohwt;TCL1NTg20.12 ± 0.02 (10)0.28 ± 0.07 (8)0.15 ± 0.04 (9)0.08 ± 0.02 (9)30.33 ± 0.09 (11)0.22 ± 0.06 (8)0.07 ± 0.03 (8)*0.07 ± 0.01 (10)40.83 ± 0.38 (11)0.57 ± 0.20 (18)0.11 ± 0.05 (16)*0.08 ± 0.01 (6)53.32 ± 2.27 (6)1.35 ± 0.46 (17)0.26 ± 0.06 (15)*0.11 ± 0.03 (5)64.38 ± 2.41 (7)5.01 ± 3.01 (9)0.66 ± 0.23 (8)0.07 ± 0.02 (5)Mean ± SEM (x1000/ul); () animals analyzed; * p<0.05 in comparison to Rhohwt; TCL1NTG (data collection still in progress)When mice from each genotype were sacrificed at 6 months of age the number of IgM+/CD5+ B-cells detected in Rhoh−/− TCL1Tg vs RhohwtTCL1Tg mice was 6-fold reduced in the peritoneal cavity, 3-fold reduced in the bone marrow and a 2-fold reduced in the spleen. In summary our data strongly suggest that RhoH plays a role in the progression of the B-CLL-like disease in the transgenic Eμ-TCL1 mouse model. This will allow detailed molecular studies of the role of RhoH in pathways altered in TCL1-induced expansion of B-cell precursor populations in this model.