P344: oncogenesis of crlf2 overexpression and effect of a novel jak2 inhibitor in crlf2 overexpressed b-cell acute lymphoblastic leukemia

Background: Cytokine receptor-like factor 2 (CRLF2), also known as TLSPR, plays an important role in the development of normal B lymphocytes, which can mediate early B cell proliferation and survival. CRLF2 overexpression caused by CRLF2 rearrangement and the gain-of-function mutation F232C has been observed in acute lymphoblastic leukemia (ALL). They are reported to contribute to oncogenesis and unfavorable outcome in ALL, particularly in Philadelphia chromosome (Ph)-like ALL. Aims: This study aimed to investigate the expression of CRLF2 and its clinical significance in adult ALL and explore the molecular mechanism of CRLF2 overexpression in vivo and in vitro. Methods: A total of 83 newly diagnosed ALL and 21 normal controls were enrolled in the cohort. Quantitative real-time PCR (qPCR) was used to detect CRLF2 expression, which was also validated by flow cytometry. CRLF2 wild type and F232C mutant was expressed in Nalm6 and 697 B-ALL cell lines with lentiviral approaches and the CCK-8 cell proliferation and colony-forming assay were performed in the cells upon the treatment of the JAK2 inhibitor, BBT594. A xenograft mouse model was generated by tail vein injection of the cells into NSG mice, and the leukemia engraftment once a week by living imaging. CRLF2 expression in ALL and its association with survival was further analyzed in Oncomine, GEPIA, cBioPortal, and TCGA database. Results: Expression of CRLF2 was significantly higher in ALL patient cohorts than that of normal control (Fig1. A). Patients with CRLF2 high expression had a significantly higher WBC count and positive rate of CD33 and CD3. Survival analysis showed that patients with CRLF2 high expression had significantly worse EFS and OS (Fig1. B-C). A significantly shorter OS and EFS were also found in the public databases. Both cell proliferation and colony-forming assay showed that CRLF2 WT or CRLF2 F232C group significantly promoted cell proliferation and colony forming in both Nalm6 and 697 cells compared to vector only group (P<0.001) (Fig1. D-F). The human leukemia xenograft mouse model showed both Nalm6-CRLF2 and Nalm6-F232C cells had a stronger ability to invasion and metastasis in the bone marrow, central nervous system, liver, and spleen of the mice. The signal intensity of the leukemia engraftment was 5-fold higher in engrafted Nalm6-CRLF2 and Nalm6-F232C cells than that of Nalm6-vector control (Fig1. G). Treatment of JAK2 inhibitor, BBT594 showed a significant dose-dependent cell proliferation arrest and clonogenicity inhibition in both Nalm6 and 697 cells expressed with either CRLF2 or CRLF2 F232C (Fig1. H-I). Summary/Conclusion: Patients with CRLF2 high expression had a worse prognosis. In vivo and in vitro data provided direct evidence of the oncogenic role of CRLF2 overexpression and the new therapeutic potential for targeting CRLF2 overexpressed B-ALL with the JAK2 inhibitor.Figure 1. CRLF2 overexpression had a oncogenesis effect in ALL. (A) The expression of CRLF2 in ALL patients was higher than normal control. (B-C) Patients with CRLF2 overexpression had a worse OS and EFS. (D-E) CRLF2 and F232C significantly promoted cell proliferation both in Nalm6 and 697 cells. (F) CRLF2 and F232C group showed a stronger clonogenicity ability than the vector-only control both in Nalm6 and 697 cells. (G) A stronger leukemogenesis and infiltration was found in CRLF2 overexpression mice. (H) A significant dose-dependent cell proliferation arrest and clonogenicity inhibition was found in CRLF2 or CRLF2 F232C overexpressed Nalm6 and 697 cells. (I) Colony numbers significantly decreased in CRLF2 overexpression cells after treated with 0.5uM BBT594 in Nalm6 cells. Keywords: Prognosis, Tumorigenesis, Gene expression, Acute lymphoblastic leukemia