DNA Damage Response Is a Barrier in the Mixed Lineage Leukemia Development.

Abstract 2399

Poster Board II-376

Mixed Lineage Leukemia (MLL)-rearranged leukemias represent a unique subset of acute leukemias that are of interest due to their distinct biological and clinical features. Although the molecular pathogenesis of the MLL-rearranged leukemias has been extensively studied, the molecular events underlying the evolution of leukemia remain to be fully understood.

We studied early changes initiated by the MLL-ENL oncogene in hematopoiesis using a MLL-ENL-ERtm knock-in mouse model wherein the protein function of the MLL-ENL-ERtm oncogene is dependent on tamoxifen. We showed that MLL-ENL, when expressed at physiologic level in all cell contexts, induces myeloproliferation both of bone marrow and splenic origin with tissue-specific hematopoietic characteristics. In mice showing an early disease phenotype, differential counts revealed a significant increase in band neutrophil population with a left shift in granulopoiesis, increase in monocytes and erythroid cells in the spleen, whereas we observed a preferential increase in band neutrophils and reduction in the erythroid compartment in the bone marrow compared to matched controls. Methylcellulose-based colony assays of MLL-ENL-ERtm spleen cells showed an increased survival and proliferation of CFU-GM, CFU-G and CFU-E progenitors compared to matched controls. In contrast, there were no marked differences in myeloid colony numbers in the MLL-ENL-ERtm bone marow at early disease state, but we observed an apparent decrease in size (cellularity) of CFU-GM colonies compared to matched controls in mice with progressed phenotype. Consistently with these observations, there was an increase in the proliferation rate of total spleen cells as measured by BrdU-incorporation assay in vivo, as well as in number of BrDU+ cells in the Mac-1+ compartment. We also detected BrdU-positivity in the Mac-1+ neutrophil compartment in the bone marrow in some animals with early disease phenotype, but interestingly we observed decline in BrDU-incorporation in the context of total bone marrow during the disease progression. These collective findings led us to hypothesize that MLL-ENL-ERtm oncogene reactivates self-renewal/proliferation in committed progenitors as it has been previously suggested by others (Krivtsov et al., Nature 2006; 442(7104):818-22), and that aberrant cell proliferation could result in activation of an intrinsic tumor suppressor mechanism. We detected expression of senescence-associated beta-galactosidase in the MLL-ENL-ERtm bone marrow, which was restricted to the neutrophil compartment and correlated with decline in the proliferation capacity of neutrophils and with upregulation of cyclin-dependent kinase inhibitors p16 and p21. Increasing rate of senescence and upregulation of p21 was evident in the spleen as well, and was maintained paralelly with increased proliferation until the late disease state. Correspondingly, the MLL-ENL-ERtm bone marrow displayed apoptosis dependently on MLL-ENL-ERtm activity, assayed by TdT-mediated dUTP Nick end labelling (TUNEL), but there was no striking difference in TUNEL positive cells in the MLL-ENL-ERtm spleen compared to matched control. Recent reports have implicated the DNA damage response (DDR) pathway in oncogene-induced senescence (Bartkova et al., Nature 2006; 444(7119):633-7), so we investigated the potential activation of DDR in response to MLL-ENL-ERtm-mediated aberrant cell proliferation. We detected the activated form of the ATM- and Rad3-related kinase (ATR, pS428), p53 phosphorylation on serine 15 and phosphorylated histone H2AX on serine 139 (γH2AX) in bone marrow cells immortalized by the activated MLL-ENL-ERtm oncogene in vitro. We found γH2AX activated in a significant number of Mac-1+ cells both in bone marrow and spleen in MLL-ENL-ERtm mice in vivo that correlated with the presence of cells undergoing senescence. Taken together, these findings indicate that MLL-ENL-ERtm-initiated aberrant cell proliferation/self-renewal in committed progenitors is sensed by DDR and coupled to senescence. Consequently, cellular senescence counteracts MLL-ENL-ERtm-mediated leukemogenesis in a setting of our disease model. Investigations are ongoing to dissect a possible mechanism that might contribute to MLL-ENL-ERtm-induced anti-cancer barrier. Grant support: MSM6198959205 and NPV2B06077.