Enhanced apoptosis of Ba/F3-ITD and 32D/ITD cells treated with FLT3 inhibitors at serial concentrations in the presence of AZD1208.

The USP9X inhibitor WP1130 enhances induction of apoptosis of Ba/F3-ITD and MV4-11 cells by quizartinib in a concentration-dependent manner.

Pim kinase and FLT3 inhibition does not enhance induction of apoptosis of cells with FLT3-WT.

AZD1208 and quizartinib co-treatment does not increase celluar ROS generation, but increases mitochondrial ROS generation.

PDF file - 141K, Table S1. Primers used for quantitative PCR analysis. Table S2. Antibodies (all diluted 1:1000) used for immunohistochemistry and Western Blot analysis.

PDF file - 1978K, Supplementary Figure 1: The gpA33 locus confers expression of tamoxifen-activatable CreERT2 transgene to the metaplastic epithelium of gastric tumors of gp130F/F mice. Supplementary Figure 2: AZD1480-treatment reduces gastric tumour burden in gp130F/F mice. Supplementary Figure 3: Immunohistochemical analysis of gastric tumors from AZD1480-treated gp130F/F mice. Supplementary Figure 4: Systemic AZD1480 administration reduces established colitis-associated colon cancer burden in wild-type mice.

PDF - 295K, Suppl. Fig. 1. Autocrine IL-6 induces metastatic colonization of CWR22Rv1 human PC cells in the livers of mice in an experimental metastases assay. Figure 2. IL-6 induces a migratory phenotype in CWR22Rv1 PC cells. Suppl. Fig. 3. Pharmacological Jak1/2 inhibitor AZD1480 suppresses IL-6-activated Stat3 signaling in CWR22Rv1 PC cells and IL-6-induced migratory PC cell phenotype.

Post-transcriptional regulation of Mcl-1 expression.

AZD1208 and quizartinib co-treatment was well tolerated in the orthotopic model.

Supplementary figure legends

Combined AZD1208 and quizartinib treatment does not abrogate growth of KG-1a cells, expressing FLT3-WT, in a xenograft model.

Quizartinib, sorafenib, crenolanib and gilteritinib cytotoxicity in cell lines with FLT3-ITD or FLT3-WT

PDF file, 966K, Supplementary Figure 1. The growth of CWR22Pc cells is down-regulated by bicalutamide. ; Supplementary Figure 2. Stat5a/b, but not Stat3, is persistently activated in CWR22Rv1 and CWR22Pc cells. Supplementary Figure 3. Inhibition of Stat5a/b, but not Stat3, significantly decreases the fraction of viable CWR22Rv1 and CWR22Pc cells. ; Supplementary Figure 4. AZD1480 induces caspase-3 activity to the same extent as genetic knockdown of Jak2 or Stat5 by shRNA. ; Supplementary Figure 5. AZD1480-treated mice maintain higher body weights than docetaxeltreated mice. ; Supplementary Figure 6. AZD1480 does not affect levels of nuclear Stat3 in CWR22Pc tumors. Supplementary Figure 7. AZD1480 induces apoptotic cell death in CWR22Pc xenograft tumors. ; Supplementary Figure 8. AZD1480 down-regulates Stat5-target gene Bcl-Xl expression in CWR22Rv1 cells in culture. ; Supplementary Figure 9. AZD1480 regulates the expression of Stat5 target genes Bcl-Xl and Ecadherin in CWR22Pc xenograft tumors.

Abstract Purpose: fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is present in 30% of acute myeloid leukemia (AML), and these patients have short disease-free survival. FLT3 inhibitors have limited and transient clinical activity, and concurrent treatment with inhibitors of parallel or downstream signaling may improve responses. The oncogenic serine/threonine kinase Pim-1 is upregulated downstream of FLT3-ITD and also promotes its signaling in a positive feedback loop, suggesting benefit of combined Pim and FLT3 inhibition. Experimental Design: Combinations of clinically active Pim and FLT3 inhibitors were studied in vitro and in vivo. Results: Concurrent treatment with the pan-Pim inhibitor AZD1208 and FLT3 inhibitors at clinically applicable concentrations abrogated in vitro growth of FLT3-ITD, but not wild-type FLT3 (FLT3-WT), cell lines. AZD1208 cotreatment increased FLT3 inhibitor–induced apoptosis of FLT3-ITD, but not FLT3-WT, cells measured by sub-G1 fraction, annexin V labeling, mitochondrial membrane potential, and PARP and caspase-3 cleavage. Concurrent treatment with AZD1208 and the FLT3 inhibitor quizartinib decreased growth of MV4-11 cells, with FLT3-ITD, in mouse xenografts, and prolonged survival, enhanced apoptosis of FLT3-ITD primary AML blasts, but not FLT3-WT blasts or remission marrow cells, and decreased FLT3-ITD AML blast colony formation. Mechanistically, AZD1208 and quizartinib cotreatment decreased expression of the antiapoptotic protein Mcl-1. Decrease in Mcl-1 protein expression was abrogated by treatment with the proteasome inhibitor MG132, and was preceded by downregulation of the Mcl-1 deubiquitinase USP9X, a novel mechanism of Mcl-1 regulation in AML. Conclusions: The data support clinical testing of Pim and FLT3 inhibitor combination therapy for FLT3-ITD AML. Clin Cancer Res; 24(1); 234–47. ©2017 AACR.

Janus kinases (JAKs) have been demonstrated to be critical in cytokine signaling and have thus been implicated in both cancer and inflammatory diseases. The JAK family consists of four highly homologous members: JAK1-3 and TYK2. The development of small-molecule inhibitors that are selective for a specific family member would represent highly desirable tools for deconvoluting the intricacies of JAK family biology. Herein, we report the discovery of a potent JAK1 inhibitor, 24, which displays, similar to 1000-fold selectivity over the other highly homologous JAK family members (determined by biochemical assays), while also possessing good selectivity over other kinases (determined by panel screening). Moreover, this compound was demonstrated to be orally bioavailable and possesses acceptable pharmacokinetic parameters. In an in vivo study, the compound was observed to dose dependently modulate the phosphorylation of STAT3 (a downstream marker of JAK1 inhibition).

Abstract AKT and Pim kinases modulate programmed cell death by the phosphorylation of common substrates that regulate apoptosis and other survival processes. Evidence suggests that the antitumor effects of targeted Akt inhibition can be inhibited or diminished by the JAK/STAT-dependent induction of Pim kinases. In this study we examined the therapeutic potential of co-targeting AKT and Pim in a genetically engineered mouse model of prostate cancer driven by the conditional inactivation of PTEN. The antitumor effects of AZD5363, a pan AKT inhibitor, and AZD1208, a highly potent Pim kinase inhibitor, were investigated as monotherapy or in combination on mice harboring castration-naïve prostate tumors and mice that developed castration-resistant disease. Mice were randomized treated for four weeks. Safety and tolerability was assessed by bodyweight changes. Antitumor activity was determined by differences in tumor burden, proliferation and apoptosis and histology. Molecular activity was assessed by examining the phosphorylation of common substrates by western blot analysis. Treatments were well-tolerated and no significant differences in bodyweight changes were observed. In castration-naïve prostate tumors, treatments with AZD5363, AZD1208 and AZD5363/AZD1208 resulted in 11.9%, 13.5% and 36.9% reductions of tumor burden compared to vehicle treated controls, respectively, P<0.001. The treatment combination of AZD5363/AZD1208 demonstrated significant antitumor activity compared to monotherapy, P<0.001. In the castration-resistant tumors, treatments with AZD5363, AZD1208 and AZD5363/AZD1208 resulted in 21%, 9.5% and 27% reductions of tumor burden compared to vehicle treated controls, respectively, P<0.004. Although the differences in in tumor burden were not statistically between monotherapy and combination therapy, a notable degree of tumor gland regression was observed in tumors treated with AZD5363/AZD1208. Overall, combination therapy showed a synergistic effect by inhibited or impaired phosphorylation of PRAS40, eIF4B and BAD in both castration-naïve and castration-resistant tumor models. Inhibition of both pathways enhanced the reduction of tumor cell proliferation and increased apoptosis. Overall, our findings provide in vivo data to support redundancy between AKT/Pim survival pathways and suggest that a therapeutic approach of combined AKT/Pim kinase inhibition may be possible therapeutic approach for AKT-driven prostate cancer. Citation Format: Marco A. De Velasco, Koichi Sugimoto, Yurie Kura, Naomi Ando, Noriko Sato, Kazuko Sakai, Barry R. Davies, Dennis Huszar, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. Co-targeting of AKT and Pim kinases in mouse PTEN-deficient prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1096. doi:10.1158/1538-7445.AM2017-1096