RPMI-8402

The Constitutive Expression of EPHB6 Receptor in Human T-ALL Promotes In Vitro Proliferation and In Vivo Cell Expansion

T-cell lymphoblastic acute leukemia (T-ALL) is a highly aggressive form of blood cancer marked by cellular subsets with abundant leukemia-initiating cells (LICs). Recent findings indicate that Ephrin receptors (Eph) are highly expressed in cancer stem cells. Investigations revealed that EphB6 is uniquely overexpressed among the Eph family in T-ALL samples, especially in a minor yet significant subpopulation with increased LIC activity. To explore EphB6's role in T-ALL, PF382 and RPMI-8402 cells were transduced with lentivectors encoding the EPHB6 gene or an empty vector as a control. Interestingly, EPHB6 expression enhanced cell expansion (Fig. 1C) and increased cell cycling, evidenced by higher Ki67 protein levels (Fig. 1D-E) and BrdU incorporation. To verify these findings in primary T-ALLs, two clones of patient-derived xenografts (PDX) were analyzed using flow cytometry, identifying a cell cluster with high EphB6 levels (Fig. 1F). EphB6 positive and negative PDX fractions were FACS sorted and tested for leukemogenic capacity via transplantation into NSG mice (Fig. 1G). Mice with EphB6+ cells died sooner than those with EphB6- cells, confirming EphB6's link to LIC. Consistent with this, LIC frequency was higher in EphB6+ populations.

Fig. 1. The constitutive expression of EPHB6 receptor in human T-ALL promotes in vitro proliferation and in vivo cell expansion (Colucci M, Trivieri N, et al., 2023).

CircFBXW7 is Depleted in T-ALL Patients and its Silencing has Functional Impact in T-ALL In Vitro

CircRNAs have emerged as significant factors in leukemogenic mechanisms, especially in T-cell Acute Lymphoblastic Leukemia (T-ALL), showing notable dysregulation. Buratin et al. investigated circFBXW7, highly expressed in T-cells and abundant compared to its linear counterpart. They conducted a loss-of-function study in T-ALL using RPMI-8402 and ALL-SIL cell lines, both with high circFBXW7 expression (Fig. 2B). Knocking down circFBXW7, while keeping FBXW7 mRNA unchanged, led to increased cell viability and proliferation (MTT and EdU assays; Fig. 2C-D). Microarray analysis revealed 2265 differentially expressed genes, with downregulated chemokines and pro-apoptotic genes (Fig. 2E-F). Key activated pathways included cell-cycle processes and mTOR signaling due to upregulation of pro-survival genes. MYC target genes and the NOTCH signaling pathway were significantly upregulated (Fig. 2G-H), with increased MYC and NOTCH1 proteins observed (Fig. 2I), suggesting a feedback loop involving MYC (Fig. 2J).

Fig. 2. CircFBXW7 is depleted in T-ALL patients and its silencing had functional impact in T-ALL in vitro (Buratin A, Borin C, et al., 2023).