Farnesyl transferase inhibitors induce extended remissions in transgenic mice with mature B cell lymphomas

Abstract

Background: We have used a mouse model based on overexpression of c-Myc in B cells genetically engineered to be self-reactive to test the hypothesis that farnesyl transferase inhibitors (FTIs) can effectively treat mature B cell lymphomas. FTIs are undergoing clinical trials to treat both lymphoid and non-lymphoid malignancies and we wished to obtain evidence to support the inclusion of B cell lymphomas in future trials.

Results: We report that two FTIs, L-744,832 and SCH66336, blocked the growth of mature B cell lymphoma cells in vitro and in vivo. The FTI treatment affected the proliferation and survival of the transformed B cells to a greater extent than naïve B cells stimulated with antigen. In syngeneic mice transplanted with the transgenic lymphoma cells, L-744,832 treatment prevented the growth of the tumor cells and the morbidity associated with the resulting lymphoma progression. Tumors that arose from transplantation of the lymphoma cells regressed with as little as three days of treatment with L-744,832 or SCH66336. Treatment of these established lymphomas with L-744,832 for seven days led to long-term remission of the disease in approximately 25% of animals.

Conclusion: FTI treatment can block the proliferation and survival of self-reactive transformed B cells that overexpress Myc. In mice transplanted with mature B cell lymphomas, we found that FTI treatment led to regression of disease. FTIs warrant further consideration as therapeutic agents for mature B cell lymphomas and other lymphoid tumors.

Figure 1

Proliferation of mouse lymphocytes and lymphoma cells in vitro in the presence and absence of L-744,832. Splenocytes were harvested from either a BCR^HEL transgenic mouse (panels a-e) or a moribund Eμ-Myc/BCR^HEL/HEL transgenic mouse (panels f-j). The splenocytes were depleted of T cells and labeled with CFSE. Cells were cultured in RPMI (panels a, f-j) or RPMI with 2 μg/ml anti-IgM and 1 μg/ml anti-CD40 (panels b-e) and 0 – 40 μM L-744,832 were included in the culture media, as indicated. After three days, cellular proliferation was measured by flow cytometry. Cells that have proliferated measure less than 10³ fluorescence units. For comparison, panels c, d, and e show proliferation in the absence of L-744,832 as a black line.

Figure 2

Treatment of lymphoma-bearing mice with L-744,832. A. L-744,832 treatment prevents the B cell lymphoma from becoming established. Unmanipulated C57BL/6 mice (Wild-type) or mice transplanted with 10⁶ Eμ-Myc/BCR^HEL/HEL transgenic lymphoma cells (Transplant recipient) were injected intravenously with 625 μg L-744,832 in 0.25 ml PBS daily (light bars) or left untreated (black bars). After 28 days, mice were euthanized and the spleens were dissected and viable cell counts of splenocytes resuspended in RPMI were determined using a hemocytometer. Error bars represent the standard deviation calculated for all mice in each group (n = 4). B and C. L-744,832 treatment causes rapid regression of lymphomas. In a separate experiment, mice transplanted with transgenic tumor cells were allowed to develop tumors for 20 or 24 days before daily treatment with L-744,832 as above for either 7 days (L744 × 7) or 3 days (L744 × 3), respectively. Twenty eight days following the tumor transplantation the mice were euthanized. Spleens were removed from tumor recipient mice and C57BL/6 control mice and photographed. Single cell suspensions from the spleens of mice that were left untreated (black bars), treated for the final 7 days (shaded bars), or treated for the final 3 days (clear bars) were counted using a hemocytometer. Data shown are the average of 4 mice in each group and error bars represent the standard deviation.

Figure 3

Treatment of lymphoma-bearing mice with SCH66336. SCH66336 treatment for 3 days causes B cell lymphomas to regress in mice. The numbers of viable, isolated splenocytes are shown from unmanipulated C57BL/6 mice (Wild-type) or mice transplanted 18 days earlier with 10⁶ lymphoma cells from an Eμ-Myc/BCR^HEL/HEL transgenic mouse (Transplant Recipient). Mice were either left untreated (black bars), treated orally for the last 3 days with 1.56 mg SCH66336 every 12 hours (shaded bars), or treated orally for the last 3 days with vehicle alone every 12 hours (clear bar). Each group contains 4 mice and error bars represent the standard deviation for each group.

Figure 4

Flow cytometry of isolated cells from mice treated with L-744,832. Relative numbers of B- and T-cells were measured from unmanipulated C57BL/6 mice (A) or C57BL/6 mice that had received transgenic lymphoma transplants 28 days previously (B). The mice were left untreated or treated with L-744,832 for 28 days (L744 × 28) or for the final 7 days (L744 × 7). The numbers of B cells (upper left quadrant) and T cells (lower right quadrant) isolated from lymph nodes (first column), spleen (second column), or thymus (third column) were determined by flow cytometry after staining with antibodies to B220 and Thy1.2. The mean percentage and standard deviation of the B220⁺ and the Thy1.2⁺ cells are shown in the appropriate quadrant of each plot. There were 4 mice in all groups, except the L744 × 28 wild-type group, which had 2 mice.

Figure 5

Tumor presence in bone marrow is blocked by L-744,832. The presence of IgM^a positive transgenic lymphoma cells in the bone marrow was measured using flow cytometry. C57BL/6 mice were transplanted with 10⁶ tumor cells from an Eμ-Myc/BCR^HEL/HEL mouse (panels b-d) or left unmanipulated (panel a). Transplant recipient mice were left untreated (panel b) or treated with L-744,832 for either 28 days (panel c) or for the final 7 days of the experiment (panel d). Bone marrow was harvested from the femurs and tibias of wild-type mice (panel a), untreated tumor recipient mice (panel b), 28-day treated tumor recipient mice (panel c) or 7-day treated tumor recipient mice (panel d). Isolated cells were stained for IgM^a and analyzed by flow cytometry. The mean percentage and standard deviation of IgM^a positive cells are indicated above each histogram for 4 mice in each treatment group.

Figure 6

Tumor cells decrease in SCH66336-treated mice. Splenocytes were isolated and stained with antibodies to IgM^a and B220 to detect the transgenic lymphoma cells. The percent of viable splenocytes positive for both markers is shown for unmanipulated C57BL/6 mice (Wild-type) or mice transplanted with 10⁶ tumor cells 18 days earlier (Transplant recipient). Results from untreated mice are shown with black bars and from transplant-recipient mice treated with 1.56 mg SCH66336 by oral gavage twice daily for 3 days with shaded bars. The average and standard deviation for 4 mice in each group is shown.

Figure 7

Long-term remission and regression of lymphomas after L-744,832 treatment. In two separate experiments (panels A and B) mice transplanted with transgenic lymphoma cells were treated for 7 days with L-744,832 starting 21 days after transplantation (blue line) or left untreated (red line). In the first experiment (panel A), 5 mice each were treated with L-744,832 or left untreated. In the second experiment (panel B), 10 mice were treated with L-744,832 and 5 mice were untreated. Mice were then monitored for signs of lymphadenopathy and euthanized when moribund.