Critical B-lymphoid cell intrinsic role of endogenous MCL-1 in c-MYC-induced lymphomagenesis

Evasion of apoptosis is critical for tumorigenesis, and sustained survival of nascent neoplastic cells may depend upon the endogenous levels of pro-survival BCL-2 family members. Indeed, previous studies using gene-targeted mice revealed that BCL-XL, but surprisingly not BCL-2, is critical for the development of c-MYC-induced pre-B/B lymphomas. However, it remains unclear whether another pro-survival BCL-2 relative contributes to their development. MCL-1 is an intriguing candidate, because it is required for cell survival during early B-lymphocyte differentiation. It is expressed abnormally high in several types of human B-cell lymphomas and is implicated in their resistance to chemotherapy. To test the B-cell intrinsic requirement for endogenous MCL-1 in lymphoma development, we conditionally deleted Mcl-1 in B-lymphoid cells of Eμ-Myc transgenic mice. We found that MCL-1 loss in early B-lymphoid progenitors delayed MYC-driven lymphomagenesis. Moreover, the lymphomas that arose when MCL-1 levels were diminished appeared to have been selected for reduced levels of BIM and/or increased levels of BCL-XL. These results underscore the importance of MCL-1 in lymphoma development and show that alterations in the levels of other cell death regulators can compensate for deficiencies in MCL-1 expression.

evasion of apoptosis is widely thought to be essential to sustain the survival of nascent neoplastic cells and hence critical for tumorigenesis. 14,15 However, the mechanisms that protect cells undergoing neoplastic transformation from apoptosis remain incompletely understood. 2,16 Abnormalities in the BCL-2-governed apoptotic pathway or its regulators have been implicated in B-cell lymphoma development. For example, BCL-2 is overexpressed due to the t [14;18] chromosomal translocation in human follicular center B-cell lymphoma, whereas both alleles of BIM are frequently lost in mantle cell lymphoma. [17][18][19][20] Accordingly, transgenic overexpression of BCL-2 (or its relatives BCL-XL or MCL-1), or engineered loss of BIM, PUMA or BAX, can accelerate lymphomagenesis, particularly if cell cycle control is impaired, for example by enforced expression of c-MYC [21][22][23][24][25] or v-Abl. 26 Although lymphomas elicited by combined overexpression of c-MYC and BCL-2 are 'addicted to' sustained BCL-2 overexpression for continued expansion, 27 endogenous BCL-2 is dispensable for c-MYC-induced lymphomagenesis. 28 In contrast, BCL-XL proved essential for the survival of both normal and pre-leukemic B cells undergoing neoplastic transformation and its loss greatly impaired lymphoma development in Eμ-Myc transgenic mice. 29 Notably, the impaired tumor development could be overcome by concomitant loss of proapoptotic BIM. 30 However, it is still unclear whether BCL-XL is the sole prosurvival BCL-2 family member required for MYC-induced pre-B/B-lymphoma development. MCL-1 is of particular interest. Increases in MCL-1 gene copy number and concomitantly elevated MCL-1 protein are found in a substantial fraction of diverse cancer types. 31 For a few cell lines derived from such cancers, MCL-1 knockdown by RNA interference was shown to cause apoptosis, demonstrating that MCL-1 is critical for their sustained survival. 31 Similarly, acute myeloid leukemia (AML) cells driven by enforced expression of c-MYC or the MLL-ENL and MLL-AF9 fusion onco-proteins and c-MYC-or BCR-ABL-driven pre-B/B lymphomas were rapidly killed upon inducible genetic deletion or blockade of MCL-1. [32][33][34][35] MCL-1 is critical for the survival of rapidly proliferating hematopoietic progenitors 36 and non-transformed pro-B/pre-B cells, 12 the cells thought to be the origin of Eμ-Myc lymphoma. 37,38 Therefore, we examined the role of MCL-1 in pre-B/B cell lymphoma development in Eμ-Myc transgenic mice by incorporating CD19-Cre or Rag1-Cre alleles to impose Mcl-1 gene deletion exclusively in the B-lymphoid compartment. We report that there was marked selection against Mcl-1 gene loss during c-MYC-driven lymphoma development and a delay in tumor onset. Moreover, the lymphomas that arose despite successful Mcl-1 fl recombination exhibited abnormally low levels of pro-apoptotic BIM and/ or increased levels of pro-survival BCL-XL. These results show that MCL-1 is critical for c-MYC-driven pre-B/Blymphoma development, and suggest that alterations in other core components of the apoptotic machinery can compensate for a reduction in MCL-1 levels.

Results
Impact of B-cell lineage-restricted deletion of Mcl-1 on MYC-driven lymphomagenesis. To explore the impact of B cell-restricted deletion of one or both allele(s) of Mcl-1 on c-MYC-driven lymphoma development, we generated Eμ-Myc mice with one or both Mcl-1 alleles flanked by loxP sites (hereafter called Mcl-1 fl/+ or Mcl-1 fl/fl , respectively). Some cohorts also expressed the Cre recombinase selectively either from the common lymphoid progenitor stage (CLP), using a Rag-1-Cre transgene, or from the late pro-B cell stage onwards, using a CD19-Cre transgene. 39 In our Mcl-1 gene-targeted mice, recombination of the Mcl-1 fl allele subjugates a human CD4 reporter transgene to the Mcl-1 promoter/enhancer elements. Hence, human CD4 (hCD4) expression, which is readily detectable by flow cytometric analysis using fluorochrome-labeled anti-human-CD4 antibodies, serves as a reporter of Mcl-1 fl deletion. 33,40,41 We first compared the incidence and rate of pre-B/B-cell lymphoma development in Eμ-Myc, Eμ-Myc;CD19-Cre, Eμ-Myc;CD19-Cre;Mcl-1 fl/+ and Eμ-Myc;CD19-Cre;Mcl-1 fl/fl mice (Figure 1a). The lymphoma-free survival of the control mice without Mcl-1 deletion (Eμ-Myc and Eμ-Myc;CD19-Cre) was similar: median survivals of 91 days and 117 days, respectively (Mantle-Cox Log-rank test P = 0.069, Figure 1a). Autopsy on the sick, lymphoma-burdened mice revealed that the Eμ-Myc;CD19-Cre;Mcl-1 fl/fl mice (P* = 0.0172) had significantly less lymphoma cells in the blood than Eμ-Myc; CD19-Cre mice, but no such drop was found for the sick Eμ-Myc;CD19-Cre;Mcl-1 fl/+ mice. No significant differences between the genotypes appeared for spleen and lymph node weights (Figure 1b), or the numbers of erythrocytes and thrombocytes in the blood (Figure 1c).   c-MYC promotes cell growth and cell proliferation, but under conditions of stress, such as nutrient or growth factor deprivation, high c-MYC levels predispose cells to undergo apoptosis. [42][43][44] Pre-leukemic Eμ-Myc mice exhibit increased numbers of pre-B cells in their bone marrow, spleen, lymph nodes and blood, but these cells are not transformed and consequently do not form tumors when transplanted into congenic recipient mice. 37 Given that loss of one allele of Mcl-1 suffices to potently induce cell death in malignant Eμ-Myc lymphomas, 35  The lymphoma-burdened, sick Eμ-Myc;Rag-1-Cre;Mcl-1 fl/+ mice showed significantly lower lymph node weights (*P = 0.031) and lymphocyte numbers in the peripheral blood (*P = 0.046) than sick Eμ-Myc;Rag-1-Cre mice (Figures 4b  and c). No significant differences were found in the spleen weights or in the numbers of erythrocytes and thrombocytes in the blood.

Discussion
Evasion of cell death is considered an essential requirement for the development of cancer. 2 Impaired apoptosis in cancer cells (particularly in hematological malignancies) often results from deregulated expression of pro-survival or pro-apoptotic members of the BCL-2 protein family. 48 In cells undergoing neoplastic transformation, apoptosis can be triggered by stress conditions induced by newly acquired oncogenic mutations (e.g. deregulated c-MYC expression) or by limiting availability of nutrients or growth factors from the tumor microenvironment. Regardless of the trigger that activates apoptosis signaling, evasion of cell death is essential for a population of nascent neoplastic cells to expand and sub-clones to acquire additional oncogenic lesions that cooperate with the initiating oncogenic mutation(s) to promote emergence of malignant cells. 16 Although BCL-2 overexpression greatly accelerates lymphomagenesis in Eμ-Myc transgenic mice, 25 endogenous BCL-2 is dispensable for MYC-driven lymphoma development. 28 In contrast, BCL-XL was found to be essential for the survival of both normal as well as c-MYC overexpressing B-cell progenitors and its loss therefore inhibited lymphoma development in Eμ-Myc mice. 29 Here we show that MCL-1 is also critical for c-MYC-driven lymphoma development.
We employed two Cre transgenic strains to delete Mcl-1 either at the late pro-B cell (CD19-Cre 39 ) or the CLP stage (Rag-1-Cre 46,47 ). Surprisingly, we found that lymphoma development in the Eμ-Myc;CD19-Cre;Mcl-1 fl/+ and Eμ-Myc; CD19-Cre;Mcl-1 fl/fl mice was only slightly slower than in the control Eμ-Myc and Eμ-Myc;CD19-Cre mice. The difference to the Eμ-Myc mice was statistically significant but the difference to the Eμ-Myc;CD19-Cre mice was not, probably because constitutive Cre activity imposes a slight toxicity in B-lymphoid cells, as previously observed in other cell types. 49  Rag1-Cre but not CD19-Cre is expressed) is more efficient in killing incipient neoplastic cells and therefore more efficient in delaying lymphoma development compared with Mcl-1 fl deletion at the later pro-B-cell stage (when CD19-Cre expression commences). Alternatively, the Rag1-Cre transgene may simply be more efficient than the CD19-Cre transgene; the latter would therefore more readily allow escape of B-lymphoid cells that had failed to excise Mcl-1 fl .
In conclusion, our findings demonstrate that MCL-1 is critical for the survival of c-MYC overexpressing lymphomainitiating cells and hence for development of lymphoma. MCL-1 appears to be more important than BCL-XL because loss of one Mcl-1 allele substantially delayed lymphoma development in Eμ-Myc;Rag-1-Cre;Mcl-1 fl/+ mice, whereas loss of one Bclx allele had only minor impact. 29,30 Loss of BIM-restored lymphoma development in mice with an Eμ-Myc; Bclx −/− lymphoid system and many pre-B/B lymphomas that arose in Eμ-Myc;CD19-Cre;Mcl-1 fl/+ or Eμ-Myc;CD19-Cre; Mcl-1 fl/fl mice despite loss of one Mcl-1 allele appeared to have undergone selection for low levels of BIM. This suggests that BIM is the critical pro-apoptotic BH3-only protein activated in response to oncogenic stress to kill Eμ-Myc pre-leukemic B-lymphoid cells to suppress progression to malignant lymphoma. These results and the observation that loss of even a single allele of Mcl-1 efficiently kills malignant Eμ-Myc lymphoma cells 41 provide further impetus to develop MCL-1 specific inhibitors (e.g. BH3 mimetics) for cancer therapy. 50,51 Materials and methods Experimental mice. All experiments with mice were conducted according to the guidelines of The Walter and Eliza Hall Institute of Medical Research Animal Ethics Committee. Eμ-Myc transgenic mice (generated on a mixed C57BL/6xSJL background and then backcrossed for 430 generations onto a C57BL/6 background) expressing the c-Myc transgene under control of the immunoglobulin heavy chain gene enhancer Eμ have been previously reported. 52 The Mcl-1 fl mice were generated on a C57BL/6 background using C57BL/6-derived ES cells. 40 The Rag-1-Cre Ki/+46 and CD19-Cre Ki/+39 mice were generated on a mixed C57BL/6x129SV genetic background using 129SV-derived ES cells and then backcrossed onto a C57BL/6 background for 420 generations before commencement of our studies.
Genotyping. Genotyping was performed as previously reported. 36 Oligonucleotide sequences for genotyping of these alleles will be provided on request.
Analysis of lymphoma-burdened mice. Eμ-Myc transgenic mice were examined daily by animal technicians for signs of malignant disease. Mice were sacrificed when declared unwell by the animal technicians. Signs of disease included splenomegaly, lymphadenopathy, hind-limb paralysis, hunched stature, weight loss and labored breathing (indicative of lymphoma growth in the thymus). Sick mice were euthanized, tissues removed, weighed and then used for flow cytometric as well as histological analyses and tissue culture.
Statistical analysis. Kaplan-Meier mouse survival curves were generated and analyzed with GraphPad Prism (GraphPad Software Inc, La Jolla, CA, USA). Mouse cohorts were compared using the log-rank Mantel-Cox test. P-values of o0.05 were considered significant. In vitro cell survival, blood cell counts, organ weights and RNA levels were plotted and analyzed with GraphPad Prism using twotailed student's t-test comparing two groups with each other. Error bars are presented as standard error of mean ( ± s.e.m.).