Integrated Analysis Of CRLF2 Signaling In Acute Lymphoblastic Leukemia Identifies Polo-Like Kinase 1 As a Therapeutic Target

Acute lymphoblastic leukemia is the most common cancer in children, and the five-year survival rate among patients older than 20 years old is still poor. Recently, Cytokine Receptor-Like Factor 2 (CRLF2) was identified as a prognostic factor in 15% of precursor B cell-acute lymphoblastic leukemia (B-ALL) that lack typical gene rearrangements (Yoda et al., Proc Natl Acad Sci U S A. 2010). In addition, several independent studies also demonstrated that CRLF2 acts as an oncoprotein (Mullighan et al., Nat Genet. 2009; Russell et al., Blood. 2009; Chapiro et al., Leukemia. 2010). The Children’s Oncology Group also found that high expression of CRLF2 confers an adverse prognosis in high-risk B-ALL patients (Chen et al., Blood. 2012). The mechanism of CRLF2-associated leukemogenesis, however, is still not clear. In CRLF2-rearranged B-ALL cases, CRLF2 is overexpressed because of genetic rearrangements. Interestingly, one-half of cases co-harbor an activating mutation in exon 16 of JAK2, most commonly involving R683 or I682. In a murine progenitor B cell line, BaF3, it has been shown that the combination of CRLF2 overexpression and JAK2 R683G confers IL-3-independent growth. This illustrates that CRLF2 overexpression could synergize with JAK2 R683G to trigger leukemic proliferation. In our previous phosphoproteomics analysis of BaF3 cells expressing CRLF and JAK2 R683G, the combination upregulated phosphorylation of Protein phosphatase 1, regulatory subunit 12A (Ppp1r12a) (van Bodegom et al., Blood. 2012), which is a negative regulator of Polo-like kinase 1 (Plk1) (Yamashiro et al., Dev Cell. 2008). In this study, we aimed to focus on novel approaches to target CRLF2 signaling in B-ALL.

To identify effective kinase inhibitors against leukemic CRLF2 signaling, we subjected BaF3 cells with CRLF2 overexpression and JAK2 R683G (BJC cells) to a kinase inhibitor screening system that consisted of 73 kinase inhibitors and other small molecules. An in vitro cell proliferation assay was coupled to observe growth inhibition at 37°C for 3 days (Tyner et al., Cancer Res. 2012). To validate ex vivo efficacy of the Plk1 inhibitor (BI-2536), which scored in the screen, we injected BJC cells with/without pre-treatment for 24 hours at 100 nM into the NOD-scid IL2Rγ^null (NSG) mice and compared spleen/liver sizes 15 days after injection. To compare in vitro efficacy of Plk1 inhibitor in CRLF2-rearranged B-ALL xenografts, we used human hTERT-transformed bone marrow stromal cells as a feeder layer and green fluorescent dye (488 nm) viability staining for flow cytometry analysis 48 hours after treatment. To investigate the downstream effect of dysregulated Plk1 in BJC cells, morphology of centrosomes was examined using anti-g-tubulin immunofluorescence staining.

The kinase inhibitor screening showed that the Plk1 inhibitor BI-2536 is particularly potent against BJC cells with an IC50 of 11 nM. To validate this screening result and test the efficacy of BI-2536 ex vivo, NSG mouse transplantation experiments showed that BJC cells that were pretreated with BI-2536 had significantly smaller spleen (0.47% vs. 1.45% of total body weight, p<0.01) and liver (5.53% vs. 7.62% of total body weight, p<0.01) sizes compared with BJC cells without treatment. The efficacy of BI-2536 against human CRLF2-rearranged B-ALL xenografts was evaluated in vitro. In comparison with vehicle control, the viability of CRLF2-rearranged B-ALL cells treated with BI-2536 was lower (48.8% of vehicle) than a wild-type CRLF2 B-ALL with BI-2536 (105.3% of vehicle). Because Plk1 is a major regulator of centrosome, we carried out immunofluorescence staining of γ-tubulin, a specific marker for centrosomes. While morphology of centrosomes showed normal patterns in BaF3 cells, in cells treated with BI-2536, replicated centrosomes failed to part from each other, surrounded by M phase chromosomes.

By integrating results from prior phosphoproteomics analysis, kinase inhibitor screening, ex vivo therapy in mouse transplantation experiments and in vitro viability assay using human xenografts, Plk1 inhibitor BI-2536 was shown to have potent activity against CRLF2-rearranged B-ALL cells. Our result suggests future therapy for this subset of B-ALL could be directed against Plk1 and its associated signaling pathways.

Tyner:Incyte Corporation: Research Funding.