Telomere Shortening By Terc Knockout in the Eµ-TCL1 Transgenic Murine Model of CLL: Characterization of Disease Development and Survival

In chronic lymphocytic leukemia (CLL) short telomeres are associated with other adverse prognostic factors and poor survival. We and others have described association of telomere length with genomic complexity and clonal evolution in CLL. To understand if telomere shortening rather than being only a marker of cell proliferation, could also functionally contribute to disease progression, we generated Terc knock out in the Eµ-TCL1 murine CLL model (Eµ-TCL1 mTerc-/- mice). Comparison of the Eµ-TCL1 mTerc-/- mice from the generations G1, G2 and G3 with that of Eµ-TCL1 (TCL1+) did not show a difference in disease initiation, progression as well as survival even though a significant decrease in telomere length of tumor cells was observed with increasing generations.

Of interest, the Eµ-TCL1 mTerc-/- G3 tumors (n=8; G3) more frequently showed defective DNA damage response compared to TCL1+ tumors (n=8), as analysed by changes in phosphorylation of gamma-H2AX, ATM and p53 measured by FACS at 1, 3, 6, 16 and 24 hours after gamma irradiation. Despite this predisposition to undergo genomic complexity, the G3 mice showed no difference in disease development compared to TCL1+ mice. Therefore we investigated if cell extrinsic factors in Terc-/- mice could affect tumor development. The Terc-/- microenvironment is known to be restrictive to B and T-lymphopoiesis and could hence inhibit CLL development. To study the impact of Terc-/- microenvironment, splenic tumors from the Eµ-TCL1 mTerc-/- G3 (n=11; G3) mice were transferred into syngeneic Terc+/+ C57Bl6 mice and compared with that of TCL1+ (n=11). No significant difference in disease burden and survival was observed between these 2 cohorts, indicating that there is no adverse influence of the Terc-/- microenvironment on the tumor growth in the Eµ-TCL1 mTerc-/- mice.

Further, we hypothesized that the telomere length in the G3 mice may not be short enough to induce genomic instability and enhance disease aggressiveness. The mice were thus crossed to obtain the Eµ-TCL1 mTerc-/- generation G4 (G4). Though the survival of these mice were similar to that of Eµ-TCL1 controls (median survival :49 vs. 51 weeks; P=0.301), the G4 mice showed significantly decreased disease burden as measured by spleen weight, liver weights and tumor cell fraction. The G4 mice showed decreased fertility and hence further crosses to generate G5 were not performed. However, further shortening of telomeres was achieved by serially transplanting the tumors from G3 into syngeneic recipient mice for 2 generations. Telomere length of the tumor cells analysed by Q-PCR showed a significant decrease with increasing transfer rounds, compared to primary TCL1+ tumors. Interestingly, the 2^nd round transfer of the G3 tumors led to a less severe disease and significantly longer survival of the recipient mice compared to 2^nd round TCL1+ tumors transplants (median survival from date of transplantation: 15 vs. 7 weeks; P=0.030), indicating that cell intrinsic factors in the G3 tumors hamper proliferation of these cells.

We finally analyzed if absence of telomerase could mask disease aggressiveness of the G3 tumors by crossing the G3 mice with wildtype Terc+/+ mice. The resulting TCL1+ mTerc+/- G4 mice showed an increased telomerase activity but had significantly shorter telomere length compared to TCL1+ mice. Strikingly, the TCL1+ mTerc+/- G4 mice showed faster disease development and significantly shorter survival (median survival: 42 vs. 51 weeks; p<0.001) as compared to TCL1+ mice. The aggressiveness of the TCL1+ mTerc+/- G4 tumors was further verified using adoptive transfer into syngeneic mice, and mice transplanted with these tumors showed a significantly shorter survival as compared to TCL1+ or G3 tumors.

In summary, the TCL1+ mTerc-/- mice crossed through generations G1 to G4 did not show a difference in disease initiation, progression or survival despite significant shortening of telomeres. However, the tumors from G3 had defective DDR, indicating a potential for accumulating genetic aberrations and clonal evolution. But the absence of functional telomerase decreased the growth potential of these genomic instable tumors. Reconstitution of telomerase in G3 mice resulted in aggressive tumors with short telomere length and could therefore be a valuable murine model for genomic instability and aggressive CLL.