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Abstract Background. The presence of chemotherapy-resistant cells can be detected in the bone marrow (BM) and peripheral blood (PB) and is called minimal residual disease (MRD). The exact mechanism for cell adhesion-mediated drug resistance leading to MRD and how to therapeutically target MRD is unsolved.Integrin alpha-6 (alpha6) is expressed on normal hematopoietic cells but has recently been described as a novel marker for MRD+ ALL cells. We previously described a role of alpha6 as a critical molecule in drug resistance of ALL. Here we extend our studies evaluating a novel non-humanized antibody targeting alpha6, P5G10, as a novel therapy against drug resistant ALL. Method. For in vitro studies patient-derived (primary) pre-B ALL cells co-cultured with murine calvaria-derived mesenchymal stromal (OP9) cells or counter-ligand laminin-1. Annexin V/7-AAD staining was used for viability determination by flow cytometry. A NOD/SCID IL2Rγ-/- (NSG) xenograft model of primary pre-B ALL was used for in vivo experiments. Results. We evaluated integrin alpha6 blockade in four primary ALL cells (LAX7R, PDX2, TXL3, SFO2) using an anti-functional alpha6 antibody, P5G10, with and without the counter ligand laminin-1 or OP9. Alpha6 blockade de-adhered all four cases from laminin-1 compared to control-treated cells and percentage of adherence was significantly different (3.3%±0.6% vs. 77.7%±3.3%, p= 0.0002 for LAX7R; 10.5%±4.9% vs. 72.5%±0.7%, p= 0.003 for PDX2; 2.0%±1.3% vs. 66.9.6%±2.6%, p=0.0002 for TXL3; 9.6%±2.8% vs. 68.0%±5.7%, p=0.0006 for SFO2).. P5G10 de-adhered leukemia cells to a lesser degree from OP9-coated plates indicating that other adhesion molecules also contribute to leukemia cell adhesion. To determine the effect of alpha6 modulation in chemoresistant ALL, primary BCR-ABL1- ALL cells (LAX7R, SFO2) were treated with Vincristine, Dexamethasone and L-Asparaginase (VDL) and BCR-ABL1+ ALL cells (TXL3, PDX2) were treated with a tyrosine kinase inhibitor (TKI), Nilotinib (NTB). Primary ALL cells showed decreased viability after monotreatment with P5G10 in a short-term assay of 2 days with laminin-1 and were sensitized when P5G10 was combined with VDL or TKI, compared to TKI monotreatment (Cell viabilities were as follows: LAX7R, 13.9%±0.6% vs. 28.8%±2.6%, p=0.009; PDX2, 12.7%±1.4% vs. 19.9%±1.5%, p= 0.037: TXL3, 32.9%±2.6% vs. 48.3%±2.5%, p=0.026; and SFO2, 34.4%±7.9% vs. 47.6%±0.1%, p=0.047). Critically, in a long-term co-culture assay of primary ALL cells with OP9 cells, alpha6 blockade in combination with VDL or NTB lead to marked decrease in viability of ALL cells compared to VDL or NTB treatment (26.5%±10.0% vs. 74.2%±2.7%, p=0.002 for LAX7R and 33.5%±11.4% vs. 84.9%±15.1%, p=0.031 for SFO2 on day 17 post treatment, respectively). To determine if P5G10 induces mobilization of leukemia cells to the peripheral blood, patient-derived ALL cells, 3 cases (TXL3, PDX2 and LAX7R) were injected into NSG mice. After determination of engraftment of leukemia by flow cytometry of human CD45 in the PB, recipient mice were treated with 30mg/kg P5G10 or PBS control by i.v. or i.p. injection. The % of human CD45+ and CD19+ in peripheral blood (PB) was analyzed by flow cytometry before (pre), 1 and 3 days after (post) treatment with P5G10. In all 3 cases, we did not observe an increase of leukemia cells in the PB compared to before P5G10 treatment (Day 0) or compared to the control recipient mice. Critically, we determined, if P5G10 can restore chemosensitivity of leukemia cells in vivo. For this purpose, we injected luciferase-labeled LAX7R cells into NOD/SCID mice. Three days after leukemia cell injection leukemia cell-bearing mice received four weekly injections of 30mg/kg P5G10 or saline ± VDL. Mice treated with P5G10 survived similarly as untreated mice (PBS: MST = 39 days vs. P5G10: MST = 31 days; p=0.05). In marked contrast, mice treated with VDL plus P5G10 survived disease-free compared to chemotherapy-only treated mice until the experiment was terminated Day 186 post-leukemia injection (MST= 185 days vs. MST=71 days; p=0.0012). Human CD19 or CD45 was undetectable in the peripheral blood by flow cytometry in surviving P5G10+VDL-treated animals before they were sacrificed. Conclusion. Taken together, we demonstrate that alpha6 may be a novel therapeutic target in ALL and modulating the function of integrin alpha6 using P5G10 can overcome drug resistance in ALL. Disclosures No relevant conflicts of interest to declare.