-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. Combinations of clinically active Pim and FLT3 inhibitors were studied and Concurrent treatment with the pan-Pim inhibitor AZD1208 and FLT3 inhibitors at clinically applicable concentrations abrogated 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-G 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. The data support clinical testing of Pim and FLT3 inhibitor combination therapy for FLT3-ITD AML. .

Fms-like tyrosine-like kinase 3 internal tandem duplication (FLT3-ITD) is present in acute myeloid leukemia (AML) in 30% of patients and is associated with short disease-free survival. FLT3 inhibitor efficacy is limited and transient but may be enhanced by multitargeting of FLT3-ITD signaling pathways. FLT3-ITD drives both STAT5-dependent transcription of oncogenic Pim-1 kinase and inactivation of the tumor-suppressor protein phosphatase 2A (PP2A), and FLT3-ITD, Pim-1, and PP2A all regulate the c-Myc oncogene. We studied mechanisms of action of cotreatment of FLT3-ITD–expressing cells with FLT3 inhibitors and PP2A-activating drugs (PADs), which are in development. PADs, including FTY720 and DT-061, enhanced FLT3 inhibitor growth suppression and apoptosis induction in FLT3-ITD–expressing cell lines and primary AML cells in vitro and MV4-11 growth suppression in vivo. PAD and FLT3 inhibitor cotreatment independently downregulated c-Myc and Pim-1 protein through enhanced proteasomal degradation. c-Myc and Pim-1 downregulation was preceded by AKT inactivation, did not occur in cells expressing myristoylated (constitutively active) AKT1, and could be induced by AKT inhibition. AKT inactivation resulted in activation of GSK-3β, and GSK-3β inhibition blocked downregulation of both c-Myc and Pim-1 by PAD and FLT3 inhibitor cotreatment. GSK-3β activation increased c-Myc proteasomal degradation through c-Myc phosphorylation on T58; infection with c-Myc with T58A substitution, preventing phosphorylation, blocked downregulation of c-Myc by PAD and FLT3 inhibitor cotreatment. GSK-3β also phosphorylated Pim-1L/Pim-1S on S95/S4. Thus, PADs enhance efficacy of FLT3 inhibitors in FLT3-ITD–expressing cells through a novel mechanism involving AKT inhibition–dependent GSK-3β–mediated increased c-Myc and Pim-1 proteasomal degradation.

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Glioblastoma multiforme (GBM) is a deadly and aggressive malignant brain cancer that is highly resistant to treatments. A particular challenge of treatment is caused by the blood–brain barrier (BBB), the relatively impermeable vasculature of the brain. The BBB prevents large molecules from entering the brain parenchyma. This protective characteristic of the BBB, however, also limits the delivery of therapeutic drugs for the treatment of brain tumors. To address this limitation, focused ultrasound (FUS) has been safely utilized to create transient openings in the BBB, allowing various high molecular weight drugs access to the brain. We performed a systematic review summarizing current research on treatment of GBMs using FUS-mediated BBB openings in in vivo mouse and rat models. The studies gathered here highlight how the treatment paradigm can allow for increased brain and tumor perfusion of drugs including chemotherapeutics, immunotherapeutics, gene therapeutics, nanoparticles, and more. Given the promising results detailed here, the aim of this review is to detail the commonly used parameters for FUS to open the BBB in rodent GBM models.

Introduction: Internal tandem duplication of the fms-like tyrosine kinase-3 receptor (FLT3-ITD) is present in acute myeloid leukemia (AML) cells in 30% of patients and these patients have short disease-free survival following chemotherapy. FLT3 inhibitors are clinically active, but their activity is limited and transient. The oncogenic serine/threonine kinase Pim-1 is transcriptionally upregulated downstream of FLT3-ITD, and promotes FLT3 signaling in a positive feedback loop in FLT3-ITD cells. We have previously demonstrated that inhibiting Pim kinase sensitizes AML cell lines and primary AML patient blasts with FLT3-ITD to induction of apoptosis by FLT3 inhibitors and by topoisomerase 2 inhibitor chemotherapy drugs. Here we studied the effects of the pan-Pim kinase inhibitor PIM447 (formerly LGH447; Novartis Pharmaceuticals), currently in clinical trials, on response of FLT3-ITD-expressing cell lines and AML patient samples to FLT3 inhibitors and to topoisomerase 2 inhibitors, with the ultimate goal of developing clinically applicable combination regimens. Methods: Cell lines and AML patient samples with FLT3-ITD were cultured with FLT3 inhibitors or topoisomerase 2 inhibitors at clinically applicable concentrations and PIM447 at a range of concentrations. Apoptosis was measured by Annexin V labeling and PARP cleavage. Mcl-1 expression was measured by immunoblotting. Reactive oxygen species (ROS) were measured with the redox-sensitive dye CM-H2DCFDA and DNA double-strand breaks (DSBs) by immunoblotting for γH2AX. Results: Transfected Ba/F3-ITD and 32D/ITD cells, MV4-11 and MOLM-14 human AML cells and primary AML patient blasts, all expressing FLT3-ITD, were treated with FLT3 inhibitors, including 100 nM midostaurin and 1 nM quizartinib, with 0, 10, 100 and 500 nM PIM447. Co-treatment with PIM447 produced a concentration-dependent increase in apoptosis induced by each FLT3 inhibitor (p<0.001). FLT3-ITD-expressing cells were also treated with the topoisomerase 2 inhibitors daunorubicin and etoposide at IC50 concentrations, with 0, 10, 100 and 500 nM PIM447. Co-treatment with PIM447 also produced a concentration-dependent increase in apoptosis induced by each topoisomerase 2 inhibitor (p<0.001). Increased apoptosis induced by PIM447 with FLT3 inhibitors was associated with Mcl-1 downregulation, while increased apoptosis induced by PIM447 with topoisomerase 2 inhibitors was associated with increased generation of ROS and increased DNA DSBs. Conclusions: The clinically applicable pan-Pim kinase inhibitor PIM447 sensitizes AML cells with FLT3-ITD to induction of apoptosis by FLT3 inhibitors and by topoisomerase 2 inhibitors. Our data support in vivo testing of combination regimens and design of clinical trials aimed at improving outcomes for patients with AML with FLT3-ITD. Citation Format: Kshama A. Doshi, Patrick R. Baldwin, Shivani Kapoor, Maria R. Baer. The clinically applicable pan-Pim kinase inhibitor PIM447 sensitizes acute myeloid leukemia cells with FLT3-ITD to induction of apoptosis by FLT3 inhibitors and by topoisomerase 2 inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3866.