Constitutive Activation of Ras and Inactivation of Ink4a/Arf Allele Induce a Fully Penetrant B-Cell Acute Lymphoblastic Leukemia Phenotype in Mice

Despite recent advances in treatment, precursor-B-cell acute lymphoblastic leukemia (B-ALL) remains a challenging clinical entity. Recent genome-wide studies have uncovered frequent genetic alterations involving activating RAS pathway mutations and loss of the INK4A/ARF locus suggesting their important role in the pathogenesis, relapse, and chemoresistance of B-ALL. Therefore, to better understand the oncogenic mechanisms by which these alterations might promote B-ALL and to develop an in vivo preclinical model of relapsed B-ALL, we engineered mouse strains with induced somatic Kras^G12D pathway activation and/or loss of Ink4a/Arf during early stages of B-cell development.

Pathologic examination revealed significantly enlarged spleens and increased numbers of mononuclear cells in the spleens of 8-week-old Kras^G12D mutated animals with or without Ink4a/Arf inactivation, but obvious histologic and IHC differences in spleens and lymph nodes between the mutant genotypes and CD19^Cre/+ controls were not found. However, in agreement with the gross pathologic examination, an increase was noted by flow cytometry in splenic B220⁺ B-cells of mice with the Kras^G12D mutation as compared with CD19^Cre/+ and CD19^Cre/+;Ink4a/Arf^L/+ mice. In keeping with this, a significant increase in number of proliferating (Ki-67⁺) cells within spleen B-cell areas of CD19^Cre/+;Kras^G12D/+, and CD19^Cre/+;Kras^G12D/+;Ink4a/Arf^L/+ mice were observed in comparison with CD19^Cre/+ controls and CD19^Cre/+;Ink4a/Arf^L/+ mice. TUNEL staining revealed fewer apoptotic cells within germinal center B-cell areas in CD19^Cre/+;Kras^G12D/+, CD19^Cre/+;Ink4a/Arf^L/+, and CD19^Cre/+;Kras^G12D/+;Ink4a/Arf^L/+ mice as compared with CD19^Cre/+ controls. Next, to identify the biological processes underlying the observed phenotypical changes, we performed gene-set enrichment analysis of microarray expression data from mutant compared to control mice. In CD19⁺ B-cells from both CD19^Cre/+;Kras^G12D/+ and CD19^Cre/+;Kras^G12D/+;Ink4a/Arf^L/+ mice, marked upregulation of gene signatures related to proliferation, Ras-related signaling pathways, apoptosis, rewired cellular metabolism, DNA repair, and pre-B stage of B-cell development were observed in both CD19^Cre/+;Kras^G12D/+ and CD19^Cre/+;Kras^G12D/+;Ink4a/Arf^L/+ mice. Consistent with our previous observations, the proliferative signature in CD19⁺ B-cells from CD19^Cre/+;Ink4a/Arf^L/+ mice was less pronounced; instead, upregulation of several signaling pathways related to independence of external growth signals and retention of "stemness" was noted. Although constitutive activation of Kras^G12D in B-cells induced prominent transcriptional changes that resulted in enhanced proliferation, it was insufficient by itself to induce development of a high-grade leukemia/lymphoma. Instead, in 40% of mice these engineered mutations promoted development of a clonal low-grade lymphoproliferative disorder resembling human extranodal marginal-zone lymphoma of mucosa-associated lymphoid tissue or lymphoplasmacytic lymphoma. Interestingly, loss of the Ink4a /Arf locus, apart from reducing the number of apoptotic B-cells broadly attenuated Kras^G12D-induced transcriptional signatures. However, combinedKras activation and Ink4a/Arf inactivation cooperated functionally to induce a fully penetrant, highly aggressive B-ALL phenotype. Cross-species bioinformatic analyses consistently demonstrated that B-ALL tumors in the CD19^Cre/+;Kras^G12D/+;Ink4a/Arf^L/+ mice recapitulated gene expression programs found in a poor-prognosis subgroups of human B-ALL that harbor BCR-ABL and CRLF2 rearrangements. Moreover, 90% of examined murine B-ALL tumors showed loss of the wild-type Ink4a/Arf locus without acquisition of common cooperating events, underscoring the role of Ink4a/Arf in restraining Kras-driven oncogenesis in the lymphoid compartment in our model.

Taken together, the functional cooperation and the negative impact on survival of concurrent Ras mutation and Ink/Arf deletion in mice suggest that these changes may also have a functional impact in human B-ALL and should be considered in the course of disease classification, as well as for prognosis and choice of therapeutic strategy.