Integrin alpha6-Deletion Induces Apoptosis and Src Signaling Changes in Acute Lymphoblastic Leukemia Cells

After induction chemotherapy, the detection of minimal residual disease (MRD) in the bone marrow of patients with acute lymphoblastic leukemia (ALL) is a negative prognostic factor. Coustan-Smith et al recently identified integrin alpha6 (Itga6) as a novel MRD marker which was included in a panel of 22 flow cytometry markers for MRD ALL cells. Our data supports a role of Itga6 as a critical molecule in drug resistance of ALL. In a xenograft model of primary pre-B-ALL, a novel non-humanized antibody targeting Itga6, P5G10, significantly prolonged survival of leukemic mice after combination with chemotherapy (Vincristine, Dexamethasone, L-Asparaginase) (n=6) compared to chemotherapy only treated mice (n=6) (MST= 185 days vs. MST=71 days; p=0.0012). In vitro treatment of primary pre-B-ALL cells adhering to laminin-1 with the anti-human Itga6 P5G10 antibody caused loss of viability, suggesting that Itga6 signaling has a pro-survival function for these cells. To investigate this further, we established murine ER^T2-Cre x Itga6^fl/fl BCR-ABL1+ pre-B ALL cells in which Itga6 function can be ablated by tamoxifen (TAM) treatment in vitro and in vivo after injection in NSG mice. In vivo ablation of Itga6 prolonged survival of recipient leukemic mice compared to Itga6 competent leukemia recipient mice. Combination of tyrosine kinase inhibition with Itga6 ablation in vivo prolonged survival significantly compared to tyrosine kinase only treated mice (MST=127 days (n=6) vs. MST of 39.5 days (n=5); p=0.0018). When ER^T2-Cre x Itga6^fl/fl B-ALL cells were treated with TAM in vitro, Itga6 expression was lost over at least 5 days. Interestingly, this corresponded with a progressive decrease in cell viability. As Src-family kinases and other tyrosine kinases play important roles in signaling of integrins, we investigated specific changes in total tyrosine phosphorylation in Itga6 deleting cells to understand the underlying mechanism for Itga6 deletion associated apoptosis. Compared to empty vector x Itga6^fl/fl pre-B ALL cells, cells losing Itga6 had increased signals on pTyr-containing proteins within 24 hours. To identify the affected proteins, we performed proteomic pTyr analysis and identified increased tyrosine phosphorylation on the docking protein CASL, which assembles signal transduction complexes including integrins, and decreases in phosphorylation of specific Y residues for Src family kinase substrates. Interestingly, increases in pY508 and pY531 in the non-receptor hematopoietic tyrosine kinases Lyn and Fyn compared to non-Itga6-deleted ALL cells were also noted. Lyn and Fyn are tyrosine residues and their phosphorylation inhibits the activity of these tyrosine kinases. Thus, kinases with increased phosphorylation on these residues have reduced tyrosine kinase activity. Treatment of Itga6^fl/fl B-ALL cells with nilotinib, to inhibit the tyrosine kinase activity of BCR/ABL1 expressed in these cells, also caused a marked increase in pY531 Fyn levels. To further address the significance of Fyn and Lyn, we treated WT and Itga6-ablated B-ALL cells with siRNA against Lyn and Fyn. Knockdown of Lyn significantly decreased viability of Itga6 WT B-ALL cells, and combined loss of Itga6 and Lyn further lowered viability. Taken together, these results implicate Lyn and Fyn with decreased viability that accompanies Itga6 deletion. Identification of Lyn and Fyn critical for transducing Itga6 signals in B-ALL will not only yield new information on the specific function Itga6, which may be useful to others who study malignancies that involve Itga6, but will also provide a rationale on which to base possible combination treatments with Itga6 antibodies.