Overcoming Drug Resistance In Acute Lymphoblastic Leukemia by Inhibition of CBP/γ-Catenin

Abstract 3264

Survivin, an inhibitor of apoptosis protein (IAP) family, has been associated with poor prognosis in cancer including leukemia. Survivin can be downregulated in colon cancer cells by inhibition of the β-catenin/Creb-binding protein (CBP) interaction using ICG-001, a small molecule specific inhibitor of the β-catenin/CBP interaction. We have shown previously that combined ICG-001 and chemotherapy can downregulate Survivin and sensitize ALL cells to chemotherapy in vitro and in a pilot study in vivo. In this study, we determine the CBP interaction with ICG-001 in primary ALL cells and preclinically evaluate ICG-001 in vitro and in vivo as an adjuvant against primary ALL and. For this purpose, primary ALL cells were co-cultured with OP9 cells and treated for 4 days with ICG-001 (10mM, 20mM) or DMSO as vehicle control. Mean viability (trypan blue exclusion) of cells treated with ICG-001 was significantly lower (ICG-001 10mM: 75.12% ± 3.15%; 20mM: 41.18%± 7.88%) compared to cells treated with DMSO (84.99% ± 0.42%) (% cell viability relative to initial control) (p=0.03). Real time RT-PCR showed ICG-001 dose-dependent downregulation of Survivin in ALL compared to control (ICG10mM vs. control: p=0.0037 and 20mM vs. control: p=0.0031). Immunoblotting demonstrated reduction of Survivin after ICG-001 treatment. Primary ALL cells incubated with a combination of VDL (Vincristine, Dexamethasone and L-Asparaginase) and ICG-001 showed decreased viability (28.7%± 4.9%) versus VDL only (79.3%± 13.6%) (p=0.014) determined by MTT assay. To elucidate if ICG-001 interacts with β-catenin/CBP as shown previously in colon cancer, we analyzed ten primary pre-B ALL cells and found significantly greater γ-catenin and Survivin expression versus normal pre-B-Cells. β-catenin was absent or in some cases expressed only weakly. Expression of v-catenin and b-catenin in ALL xenograft cells were detected by Western blot. One primary ALL was selected and incubated with γ-catenin and β-catenin siRNA for 48hrs, followed by 6hrs incubation with Wnt3a. Wnt3a induced both of γ-catenin and β-catenin expression. Survivin was reduced by γ-catenin siRNA but not β-catenin siRNA treatment. Addition of Wnt3a partially recovered the decrease of Survivin. In addition, Survivin was knocked down in primary ALL using shRNA and non-silencing shRNA control or ICG-001 (2uM) and DMSO control. Western blot analysis showed that survivin shRNA or ICG-001 treatment lead to downregulation of Survivin and γ-catenin. Using a ChIP assay we could demonstrate occupancy of TCF4 and CBP association at the Survivin promoter, which was not altered by ICG-001 in primary ALL. Moreover, ICG-001 treatment of primary ALL cells prevents CBP but not p300 occupancy. For further preclinical in vivo evaluation of ICG-001, one Philadelphia chromosome positive ALLs (Ph⁺) and two Ph⁻ primary ALL were injected into sublethally irradiated NOD/SCID IL2Rγ^−/-mice and treated with ICG-001 (50mg or 100mg/kg/day per subcutaneous miniosmotic pump) with or without chemotherapy including VDL for Ph⁻ ALL (per intraperitoneal injections) or Nilotinib for Ph⁺ ALL (per os). For analysis we pooled the survival of all three primary leukemias. The saline control group (n=10) (MST= 55.5.days) and the ICG-001 only groups (n=3) (MST=61 days) died rapidly. The group treated with chemotherapy (n=13) had a median survival time (MST) of 85 days. In marked contrast, the group treated with the combined chemotherapy+ICG-001 (n=15) lived significantly longer (MST=100) (p<0.05). Taken together, our data shows that Survivin transcription can be mediated by γ-catenin in primary ALL and that targeting CBP/γ-catenin by using ICG-001 ALL can sensitize ALL cells to chemotherapy in vitro and in vivo.