IFITM (Interferon-induced transmembrane protein) also known as CD225 was identified as a gene that are transcriptionally induced downstream of interferon (IFN) signaling. Ifitm3, basally expressed on the plasma membrane, is associated with CD19 (B cell receptor components), CD81 and CD21 in mouse B cell. However, the specific roles of Ifitm3 in B cells remain unclear.
We found that high expression levels of Ifitm1 and 3 mRNA in ALL patient samples at the time of diagnosis correlated with positive minimal residual disease (MRD) status in two clinical trials for patients with high risk acute lymphoblastic leukemia (n=207; COG P9906; p=0.005 and ECOG E2993; n=215; p=0.01). In addition, high expression levels of Ifitm3 mRNA at diagnosis predicts poor overall survival (OS) in Ph+ ALL patients (ECOG E2993; n=83; p=0.01). To study the function of Ifitm in Ph+ ALL, B cell progenitors from Ifitm3-/- mice lacking Ifitm3 were transformed with BCR-ABL1. Loss of Ifitm3 showed significant cell cycle arrest in the G0/G1 phase (42.5%, p<0.001) compared to wild-type Ph+ ALL cells (28%) through increased levels of p53, p21. Interestingly, loss of Ifitm3 also showed upregulation of AKTS473 phosphorylation and c-Myc expression, well-established markers of oncogene-induced senescence with accumulation of p53. β-galactosidase assay revealed that loss of Ifitm3 induced 2.8 fold (P=0.04) higher cellular senescence than wild-type Ph+ ALL cells. Moreover, treatment of Adriamycin (25 ng ml-1) for induction of low level of DNA damage significantly induced cellular senescence on most Ifitm3-/- Ph+ ALL cells, but had no effect on wild-type Ph+ ALL cells. Consistent with Ifitm3-mediated proliferative defects of Ph+ ALL cells, Ifitm3-/- Ph+ ALL cells exhibited reduced self-renewal capacity with 0.33 fold (P=0.0004) decreased colony in colony forming assay compared to wild-type Ph+ ALL cells. Furthermore, Ifitm3 deficient B cell progenitors also showed significant inhibition of proliferation with inhibition of phosphorylation of Stat5Y694 and c-Myc expression. We found that loss of Ifitm3 leads to impaired membrane expression of CD19 in both B cell progenitors and Ph+ ALL cells. 4-hydroxytamoxifen (4-OHT)-inducible activation of CD19 enhanced the proliferation of wild-type Ph+ ALL cells and completely rescued proliferative defects in Ifitm3 deficient B cell progenitors with release from G0/G1 cell cycle arrest associated with upregulation of phosphorylation of Stat5 Y694 and c-Myc expression. Phosphorylation of BTKY223 known as downstream effector of CD19 was also induced by forced expression of CD19 in both wild-type and Ifitm3 deficient B cell progenitors. In addition, we found that CD19 positively regulates the surface expression of IL7R in B cell progenitors. Interestingly, CD19low population in Ifitm3 deficient B cell progenitors showed lower surface expression of IL7R compared to CD19high population. In contrast to B cell progenitors, forced expression of CD19 did not increase the proliferation of wild-type Ph+ ALL cells. Surprisingly, upregulation of CD19 induced apoptosis in Ifitm3-deficient Ph+ ALL cells with significant inhibition of BCR-ABL1 activity, phosphorylation of Stat5Y694, BTKY223and Bcl2 expression.
These findings identify novel role of Ifitm3 in both B cell progenitors and Ph+ ALL cells. Ifitm3 regulates the dual function of CD19 as a positive regulator of IL7R, which mediates the proliferation of B cell progenitors and as a negative regulator, limiting the activity of BCR-ABL1 in Ph+ ALL.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.