As described previously (20,24-26) bone marrow cells were obtained from patients with B-ALL after informed consent was obtained under Institutional Review Board of Children's Hospital Los Angeles approved protocols. Primary B-ALL blasts were obtained from the bone marrow aspirates by separation using Ficoll (Cytiva) gradient centrifugation of 450 x g at 20˚C for 30 min then pellets were harvested. Harvested mononuclear cells (0.01-1x10⁶/200 µl PBS/mouse) were intravenously injected into NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) female mice (Jackson Laboratory; total of 129 mice; age, 5-7 weeks; weight, 17-24 g), which were conditioned with a single sub-lethal dose of 250 cGy of whole body irradiation at 21-23˚C -room temperature, and expanded. Mice had free access to food and water and were housed at 21-23˚C room temperature, at 30-70% humidity and with a 6:00 am-7:00 pm light cycle. The mice were sacrificed using CO₂ at 30% air displacement rate. Engrafted human leukemia cells were harvested from the bone marrow and spleen 2-26 weeks after injection, before they were cultured at 37˚C with 5% CO₂ further in the presence of murine stromal OP-9 cells (American Type Culture Collection, ATCC) in vitro. Primary B-ALL cells (LAX7, LAX7R, LAX53, LAX56, TXL3 and ICN24 from the Lab of Dr. Yong-Mi Kim) were co-cultured with OP-9 stromal cells in MEM-α (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 20% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin. The B cell lineage-ALL (B-ALL) cell lines were all incubated at 37˚C with 5% CO₂. TOM-1 (DSMZ) and BV173 (DSMZ) were cultured in 80% RPMI-1640 (Invitrogen; Thermo Fisher Scientific, Inc.) + 20% FBS. SupB15 (ATCC) was cultured in 80% IMDM (Invitrogen; Thermo Fisher Scientific, Inc.) + 20% FBS. REH (ATCC), RS4;11 (ATCC), Kasumi-2 (DSMZ), 697 (DSMZ), BEL1 (DSMZ), RCH (DSMZ) were in cultured in 90% RPMI 1640 + 10% FBS. Cytogenetic information and α-integrin expression percentages of all B-ALL cell lines used for the present study are shown in Table SI (25,26). Human umbilical vein endothelial cells (HUVECs; Thermo Fisher Scientific, Inc.) were cultured in Medium 200 supplemented with 0.2% LSGS (Invitrogen; Thermo Fisher Scientific, Inc.) at 37˚C with 5% CO₂.

AVA4746 and TBC3486 were obtained from Aviara Pharmaceuticals, Inc. for research use. MG132 was obtained from Selleck Chemicals. Vincristine, Dexamethasone and L-asparaginase were obtained from Children Hospital of Los Angeles.

After harvesting, B-ALL cells or HUVECs (0.1-1x10⁶) were centrifuged at 500 g for 5 min at room temperature and resuspended in 100 µl PBS containing anti-human CD19 (#302206, 1:20 dilution, Biolegend, Inc.), anti-human CD49d (#304304, 1:20 dilution, Biolegend, Inc.) and anti-human CD29 (#303008, 1:20 dilution, Biolegend, Inc.) antibodies, alongside the isotype control mouse IgG1κ (Biolegend, Inc.). Following incubation at 4˚C for 30 min, cells were washed with 1 ml PBS and resuspended in 100 µl DAPI/PBS (0.1µg/ml) for 15 min at 4˚C for assessment using a flow cytometer (BD FACSCanto II, BD Biosciences). Flow cytometry data were analyzed from the viable cells in the DAPI-negative population, based on an isotype gating strategy using Flow Jo (version 10, BD Biosciences).

For apoptosis assay, B-ALL cells were treated for 48 h with either DMSO or AVA4746 25 µM, with or without VDL chemotherapy (vincristine 5 nM, dexamethasone 0.05 nM, L-asparaginase 2.5x10^-3 IU/ml) in the presence or absence of OP9. Annexin V (#640947, 1:20; Biolegend, Inc.) and DAPI were used to stain the 1x10⁶ cells for 15 min at room temperature (25˚C), in the dark.

As previously described (27,28), LAX7R or TXL3 cells were suspended in the HEPES buffer (110 mM NaCl, 10 mM KCl, 10 mM glucose, 1 mM MgCl2, 1.5 mM CaCl2 and 30 mM HEPES, pH 7.4 containing 0.1% HSA Sigma-Aldrich; Merck KGaA) at a density of 1x10⁶ cells/ml. B-ALL cells were then incubated with either AVA4746 of 0.00, 0.01 µM, 0.1 µM, 1 µM, 10 µM, or 100 µM or human VCAM-1 (PeproTech, Inc.) of 0 µM, 1.35 µM, 13.5 µM, 135 µM, or 1350 µM in the presence of 10% phycoerythrin (PE)-conjugated HUTS-21 antibodies (#556049, BD Biosciences) with or without 5mM Mn²⁺ for 30 min at 37˚C. Subsequently, 0.5 µl DAPI (200 µg/ml) was added to each reaction 5 min before the end of the incubation time. The PE-HUTS-21 mean fluorescence intensity (MFI) was analyzed by flow cytometry (BD FACSCanto II, Flow Jo version 10, BD Biosciences). Receptor occupancy (RO) assays are designed to quantify the binding of therapeutics to their targets on a cell's surface (29). The protocol used for calculating the normalized VLA-4 RO was as follows: The maximal MFI value from highest concentration of AVA4746 or VCAM-1 was normalized as 100%, whilst the minimum MFI value from lowest concentration of AVA4746 or VCAM-1was normalized as 0%. EC50 of RO (half maximal occupancy of VLA-4 receptor concentration) was calculated using GraphPad Prism 5 software. To determine a level of non-specific binding, cells were stained in parallel with the 10% isotype control antibody (PE Mouse IgG2a, κ Isotype Control, #555574, BD Biosciences).

Cell adhesion and de-adhesion assay. For the cell adhesion assay, non-tissue culture 96-well plates were coated with 10 µg/ml human VCAM-1 (PeproTech, Inc.) overnight at 4˚C, washed and blocked with 2% BSA (Sigma-Aldrich; Merck KGaA) for 30 min at room temperature. LAX7R or TXL3 cells (1x10⁶/well) were incubated with 0.00, 0.01 µM, 0.1 µM, 1 µM, 10.00 or 100 µM AVA4746 at room temperature for 1 h, added into the plates pre-coated with VCAM-1 and were allowed to adhere for 1 h at 37˚C. Non-adherent cells were removed before being gently washed once by PBS, whilst adherent cells were counted using a hemocytometer following Trypan Blue (Invitrogen; Thermo Fisher Scientific, Inc.) exclusion.

Similarly, B-ALL cells (1x10⁶/well) were seeded into the plates pre-coated with 10 µg/ml human VCAM-1 or OP9 cells for 4 h at 37˚C in 5% CO₂. Next, either AVA4746 (25 µM) or DMSO was added as a vehicle control and the cells were incubated at 37˚C with 5% CO₂ overnight. The non-adherent cells in the supernatant were then removed, whereas alive adherent cells were detached by pipetting with PBS prior to counting by using Trypan Blue exclusion of dead cells with a hemocytometer. Finally, the percentage of adhesion was calculated by dividing the number of live adherent cells by the total number of cells.

B-ALL cells were treated with AVA4746 (0,1 µM, 5 µM, or 25 µM) for 24 h or 96 h for on-target effect experiments. Furthermore, following 2 h pre-treated with MG132 0 µM or 1 µM B-ALL cells were treated with AVA4746 either 0 µM or 25 µM for 96 h as separate experments. B-ALL cells were harvested and lysed in M-PER^™ Mammalian Protein Extraction Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) containing a 1% protease inhibitor cocktail (VWR International, LLC). Protein concentration was measured using Bradford protein assay. Protein lysates (20 µg/lane) were separated in 4-12% Bis-Tris protein gels by SDS-PAGE and transferred onto PVDF membranes. Then the membranes were blocked by 5% dry fat milk for 1 h at room temperature. The following primary antibodies were incubated overnight at 4˚C: Anti-integrin α4 (#4600, 1:1,000; Cell Signaling Technology, Inc.), anti- phosphorylated (p-)-AKTSer473 (#9271, 1:1000; Cell Signaling Technology, Inc.), anti-AKT (#9272, 1:1,000; Cell Signaling Technology, Inc.), anti-phosphotyrosine (#05-321MG, 1:1000 dilution, EMD Millipore) and anti-β-actin (#sc-47778, 1:2000 dilution, Santa Cruz Biotechnology, Inc.). Secondary antibody solution Alk-Phos. conjugated (anti-rabbit #WP20007 or anti-mouse #WP20006; Thermo Fisher Scientific, Inc.) was applied at room temperature for 1 h. To visualize bands, chemiluminescent substrate (#WP20002, Thermo Fisher Scientific, Inc.) was used. Western blots quantify was analyzed via Image J (version 1.53e, National Institutions of Health).

B-ALL cells were first serum-starved by being washed twice with Dulbecco's PBS (DPBS) and cultured in MEM-α media at 37˚C and 5% CO₂ overnight. Following another wash with DPBS, B-ALL cells were treated with either the vehicle control 0.1% DMSO or AVA4746 (5 or 25 µM) for 30 min at room temperature. Subsequently, FBS was added to a final concentration of 20% to all cells except for those in the no-activation control groups. In either the human VCAM-1 or OP9 group, 1x10⁶ cells were seeded into plates pre-coated with either human VCAM-1 (10 µg/ml) or OP9 (0.2x10⁶ cells/well on 12-well plate were pre-coated at 37˚C for 1 day before experiment). Whole cell lysates were isolated after incubation for either 1 h or 24 h at 37˚C for use in western blot analysis for either p-AKT or phosphotyrosine detection.

Mobilization of leukemia cells was tested according to a previously described protocol (20). Primary B-ALL LAX7R cells (0.05x10⁶ in 200 µl PBS per mouse) were intravenously injected into in total of 14 NSG mice (7 mice in PBS group while 7 mice in AVA4746 treatment group). In total, 80 µl peripheral blood was collected each mouse by retroorbital exsanguination following 2% isoflurane anesthesia once a week from weeks 2 to 4 after the injection of LAX7R cells. The total duration of the blood sampling protocol was typically 3-6 min. When ~0.5 or ~2.5% hCD45⁺ hCD19⁺ LAX7R cells were detected by flow cytometry in the peripheral blood (PB) of NSG mice, the mice were treated with either PBS or AVA4746 (60 mg/kg) by intraperitoneal (i.p.) injection. Following 100 µg/ml purified human IgG (#I2511, Sigma-Aldrich, Inc.) as blocking reagent for elimination of non-specific binding, 0.1x10⁶ cells were stained with APC-hCD45 (#304012, 1:20 dilution, BioLegend, Inc.), PE-hCD19(#302254, 1:20 dilution, BioLegend, Inc.), FITC-mCD45 (#103108, 1:20 dilution, BioLegend, Inc.), and APC-Cy7 (#304328, 1:20 dilution, BioLegend, Inc.) at 4˚C in dark for 30 min. Subsequently, 8 or 36 h after treatment, the mice were sacrificed using CO₂ at 30% displacement rate. Following animal sacrifice whole mononuclear cells were isolated from the PB, bone marrow (BM) and spleen (SPC) using the ACK lysing buffer (Invitrogen; Thermo Fisher Scientific, Inc.) at either 8 or 36 h after treatment. Alive mononuclear cell (MNC) numbers were counted with a hemocytometer using trypan blue exclusion, whilst the percentages of either mCD45⁺ or hCD45⁺hCD19⁺ cells were determined by flow cytometry (BD FACSCanto II, Flow Jo version 10; BD Biosciences). Therefore, number of mouse cells or leukemia cells was calculated as MNC number x percentage of mCD45⁺ or hCD45⁺hCD19⁺ for mouse and human leukemia cells, respectively. This animal study was performed in compliance with a research protocol approved by the Institutional Animal Care and Use Committee at the Saban Research Institute of Children's Hospital Los Angeles.

A Matrigel-coated 48-well plate (#354508, Biocoat^™; BD Biosciences) was first pre-warmed at 37˚C for 30 min. HUVECs (4-6x10⁴/well) that were treated with AVA4746 (0, 5 or 25 µM) or TBC3486 (0, or 25 µM) at 37˚C for 30 min were seeded into each well and incubated for 4-6 h at 37˚C in 5% CO₂. After incubation, images of three representative fields of view per well were obtained using phase contrast light microscopy (x100 magnification), before being analyzed using the ‘angiogenesis analyzer’ tool (Gilles Carpentier) in ImageJ (30) (version 1.53e, National Insititutes of Health). Tube formation was assessed by analyzing the number of nodes (pixels with ≥ 3 neighboring elements corresponding to a bifurcation), segments (elements delimited by two junctions), meshes (areas enclosed by segments or master segments and made by tube-like structures) and total area, before being quantified.

Primary B-ALL cells (0.05x10⁶) were intravenously injected into each NSG mouse. In total of 91 mice were divided into four treatment groups: PBS (5 mice in LAX7R exp#1, 4 mice in LAX7R exp#2, 7 mice in RS4;11, 5 mice in TXL3), AVA4746 group (5 mice in LAX7R exp#1, 5 mice in LAX7R exp#2, 7 mice in RS4;11, 6 mice in TXL3), VDL group (6 mice in LAX7R exp#1, 5 mice in LAX7R exp#2, 6 mice in RS4;11, 6 mice in TXL3), and VDL+AVA4746 group (6 mice in LAX7R exp#1, 5 mice in LAX7R exp#2, 8 mice in RS4;11, 5 mice in TXL3). AVA4746 (15 or 30 mg/kg dissolved in PBS) was then administered by oral gavage twice a day continuously for 14, 21 or 28 days, with treatment starting 2 days after B-ALL cell injection. Subsequently, starting at 3 days after B-ALL cell injection, vincristine (0.5 mg/kg) was administered (i.p.) once a week for 2 or 4 weeks, whilst dexamethasone (10.5 mg/kg) and L-asparaginase (1500 IU/kg) were administered (i.p.) once a day (but not at weekends) for 2 or 4 weeks. Animals were monitored daily and the survival time of the mice was then recorded. Animals were sacrificed by CO₂ euthanasia at an air displacement rate of 30% if >15% weight loss was observed or physical changes, including loss of ambulation, were observed.

Statistical significance was assessed using a two-tailed Student's t-test and the data are presented as the mean ± SD. One-way analysis of variance (ANOVA) with Tukey's post hoc test was used for comparisons of > two groups. Survival data were analyzed by Kaplan-Meier survival curves and comparisons were performed using the log-rank test for two groups without adjustment. P<0.05 was considered to indicate a statistically significant difference. All statistical analysis was performed using GraphPad Prism 5 software (GraphPad Software, Inc.). At least two of experimental repeats for in vitro assays.

Since AVA4746 is a small molecule mimetic compound of VCAM-1(23), conformation activation experiments were performed to determine the half maximal effective concentration (EC50) of AVA4746. HUTS-21 is a specific monoclonal antibody that targets an epitope corresponding to the active conformation of integrin β1(31). Anti-HUTS-21 antibody binding would occur when VLA-4 binds to certain ligands, such as VCAM-1(31). The HUTS-21 antibody binding curve represents the VLA-4 RO, which is used as an indicator of binding affinity (31). In LAX7R cells, the EC₅₀ of AVA4746 was 38.52 nM (26.84-55.28 nM) and 1357.00 nM (1295.00-1422.00 nM) in the presence (Fig. S1C) or absence of 5 mM Mn²⁺ (Fig. S1A), respectively (Fig. 1A). In TXL3 cells, the EC₅₀ of AVA4746 was 32.68 nM (24.56-43.49 nM) and 973.00 nM (914.00-1036.00 nM) in the presence (Fig. S1C) or absence of 5 mM Mn²⁺ (Fig. S1A), respectively (Fig. 1B). By contrast, the EC₅₀ of human VCAM-1 was 102.20 µM (67.41-154.90 µM) and 110.60 µM (63.81-191.90 µM) in the presence of 5 mM Mn²⁺ in LAX7R and TXL3 cells, respectively (Figs. 1C, D and S1D). There was little conformation changes corresponding to VLA-4 activation by human VCAM-1 without 5 mM Mn²⁺ (Figs. 1C, D and S1B). The MFI of PE-HUTS-21 shown in Table SII are from two independent experiments performed in triplicate. Taken together, AVA4746 exhibits nanomolar levels of affinity for the active conformation of VLA-4, which was ~3,500-fold higher compared with that of VCAM-1 in the presence of Mn²⁺.

Other soluble integrin ligands present in the serum may interfere with VLA-4 ligand binding (31), since the primary B-ALL cells were cultured in MEM-α in the presence of 20% FBS. Therefore, VCAM-1 cell adhesion assays were used to determine the half maximal inhibitory concentration (IC50) of AVA4746. The IC₅₀ of AVA4746 was 8.12 (7.07-9.32 µM) and 13.95 µM (11.15-17.45 µM) in LAX7R and TXL3 cells, respectively, in one out of the two independent experimental repeats (Fig. 1E and F). The percentages of adhesion from the two independent experiments are shown in Table SIII. To achieve a saturating competitive effect, 5 and 25 µM AVA4746 were used for subsequent experiments.

Primary B-ALL cells derived from four human patients (LAX7R, LAX7, LAX53 and LAX56) and the B-ALL cell line REH were used to evaluate the on-target effect of AVA4746 on B-ALL in vitro. Cells were treated with either AVA4746 (1, 5 and 25 µM) or 0.1% DMSO as a control for either 24 or 96 h, before they were analyzed by flow cytometry to detect human integrin α4 expression. The MFI of integrin α4 on the B-ALL cells was decreased by AVA4746 in a dose-dependent manner, whilst the MFIs of integrins α5 and α6 were not affected (Figs. 2A-E and S2). AVA4746 also caused a reduction in the protein expression levels of α4 as determined by western blot analysis, after 24 and 96 h of treatment (Fig. 2F). Subsequently, the effects of proteasomal degradation on this decrease in integrin α4 protein expression were subsequently tested. B-ALL cells were treated for 96 h with either AVA4746 (25 µM), MG132 (1 µM) or DMSO as a vehicle control, alone or in combination. α4 was partially restored by MG132 in all cases apart from LAX56 (Fig. S3.). These results suggest that this decrease in α4 expression induced by AVA4746 may also be independent of the ubiquitination-proteasome pathway. In addition, AVA4746 slightly downregulated the phosphorylation levels of AKT following stimulation with human VCAM-1 in serum-starved LAX7R cells, but no obvious changes were observed following AVA4746 treatment after stimulation with serum (Fig. S4A). Similar tendency was found in LAX56 (Fig. S4A). These results suggest that other cell signaling pathways may be involved (Fig. S4A), since AVA4746 may also affect phosphotyrosine cell signaling in LAX53 cells (Fig. S4B). Clear decreased band densities were found in both 5 µM and 25 µM AVA4746 compared with 0 µM in 1 h treatment group around 50-60 kDa.

To investigate the competing ligand effect, the de-adhesion assays were performed as previously described (21). Five types of primary B-ALL cells, namely LAX7R, LAX53, LAX56, TXL3 and ICN24 cells (Fig. 3A-E), along with seven B-ALL cell lines, namely RS4;11, SupB15, Kasumi-2, 697, BEL-1, BV173 and RCH cells (Fig. 3F-L; Table SI), were plated onto either human VCAM-1- (10 µg/ml) or 2% bovine serum albumin (BSA)-coated plates, before being treated with either DMSO or 25 µM AVA4746 overnight. All 12 types of B-ALL cells showed significant de-adhesion from the VLA-4 ligand, human VCAM-1, after AVA4746 treatment compared with that in the DMSO control (Fig. 3). In addition, AVA4746 was found to induce the detachment of eight B-ALL cell lines from the OP9 stromal cell line (Fig. S5A-H). Since the murine calvaria-derived stromal cell line OP9 has been found to facilitate survival and drug resistance in B-ALL cells (20), Annexin V assays were subsequently performed to determine the apoptotic effects of AVA4746 in combination with the chemotherapeutic regimen of 0.5 mg/kg vincristine, 10.5 mg/kg dexamethasone and 1500 IU/kg L-asparaginase (VDL). The small but significant induced more apoptosis were found in VDL+AVA4746 compared with VDL alone in TXL3 (without OP9, Fig. S6A), LAX56 (without OP9, Fig. S6B) and LAX7R (with OP9, Fig. S6C). However, neither AVA4746 alone nor AVA4746 in combination with VDL induced noTable apoptotic effects in B-ALL cells compared with that in the corresponding groups that were not treated with AVA4746, regardless of the presence of OP9 cells (Fig. S6A-C).

To determine the role of integrin α4 in B-ALL cell metastasis, mobilization assays were performed. In brief, AVA4746 treatment was initiated when detecTable B-ALL cells could be observed in PB. Subsequently, mice were sacrificed before the PB, BM and SPC were harvested for analysis. When ~2.5% LAX7R cells was detected in the PB (Fig. S7A), mice were treated with either AVA4746 (60 mg/kg; n=6), PBS as a control (n=6), VDL (n=6) or VDL + AVA4746 (n=6).

After 8 h of treatment, PB, BM and SPCs were harvested for a human leukemic population analysis using flow cytometry. There was an insignificant but decreasing tendency of leukemia (% of hCD45⁺hCD19⁺) in the PB collected from the AVA4746 group compared with that in the PBS group (Fig. S7B), whilst there was no significant difference between BM and SPCs (Fig. S7C and D). The percentage of leukemic cells (hCD45⁺hCD19⁺) was significantly decreased in both VDL and VDL + AVA4746 groups compared with that in the PBS groups, but there was no statistical difference between that in the VDL and VDL + AVA4746 groups (Fig. S7B). Additionally, a significant decrease in the human α4 MFI under the leukemic population (hCD45⁺hCD19⁺) was exclusively found in AVA4746 groups compared with that in the PBS group in the BM samples (Fig. S7C).

The timeframe of the initial treatment was subsequently optimized, so that when the leukemia cell (hCD45⁺hCD19⁺) percentage in the PB was 0.5%, the duration of AVA4746 treatment was prolonged to 36 h (Fig. 4A). Consequently, AVA4746 significantly decreased the percentage of B-ALL cells (hCD45⁺hCD19⁺)in the PB compared with that in the PBS control (Figs. 4B and S8A) whilst significantly increasing that of B-ALL cells (hCD45⁺hCD19⁺)in the BM (Figs. 4C and S8B). No changes were found in the SPC samples (Figs. 4D and S8C).

Results from the in vivo mobilization assays suggest that AVA4746 can cause the retention of B-ALL cells in the BM. Since integrin α4 and β1 were found to be expressed on endothelial cells (Fig. 5A), it was hypothesized that the underlying mechanism may be due to the AVA4746-mediated inhibition of α4 on endothelial cells. This may in turn reduce vessel formation and reduce the release of B-ALL cells into the peripheral circulation. To test this hypothesis, the potential effects of AVA4746 on HUVECs were evaluated in vitro. AVA4746 signficantly attenuated the in vitro angiogenic potential of HUVECs. Representative images of HUVECs in Fig. 5B demonstrate that the number of nodes, segments, meshes and total meshes was all significantly decreased by both 5 and 25 µM AVA4746 (Fig. 5C). Similar results were found using TBC3486, an analog of AVA4746 (Fig. S9). However, there were no statistical differences in angiogenesis inhibition using NZM (data not shown).

To further determine the effects of AVA4746 on the survival of human B-ALL cell mouse xenograft models, LAX7R cells (0.05x10⁶ cells per mouse) were injected into the NSG mice intravenously. Subsequently, 2 days post-injection, either AVA4746 or PBS was administered by oral gavage at 15 mg/kg, twice a day (30 mg/kg/day) for 14 days. VDL was given 3 days post-injection for 4 weeks by i.p. (Fig. 6A). A significant prolongation of overall survival in the group treated with a combination of AVA4746 and VDL (n=6 MST; 78.5 days) was found compared with that in the VDL-only group (n=6; MST, 68 days; Fig. 6B). By contrast, there was no difference between the PBS (n=5; MST, 38 days) and the AVA4746 groups (n=5; MST, 38 days; Fig. 6B). An increase in the AVA4746 dosage to 60 mg/kg/day and an increase in the treatment time to 28 days did not result in an improvement in survival time compared with AVA4746 30 mg/kg/day (Fig. 6C). However, significantly prolonged survival in the AVA4746 + VDL group was again observed (n=5; MST, 87 days) compared with that in the VDL-only group (n=5; MST, 77 days; Fig. 6D).

In addition, mouse xenograft models were established using cell lines derived from two other cases B-ALL (RS4;11 and TXL3). AVA4746 in combination with VDL (n=8; MST, 105 days) did not lead to a statistically significant improvement in the survival of mice injected with RS4;11 cells in the VDL-alone group (n=6; MST, 95 days; Fig. S10A and B). By contrast, AVA4746 alone (n=6; MST, 66 days) prolonged the survival of the TXL3 xenograft mice compared with that in the PBS control (n=5, MST, 59 days; Fig. S10C and D). However, there were no changes in the survival of mice in the group treated with AVA4746 + VDL compared with VDL alone in the TXL3 model.