Oncogenic FLT3 Research Articles

Disrupting PTPRJ transmembrane-mediated oligomerization counteracts oncogenic receptor tyrosine kinase FLT3 ITD.

The receptor protein tyrosine phosphatase (RPTP) PTPRJ (also known as DEP-1) has been identified as a negative regulator of the receptor tyrosine kinase FLT3 signalling in vitro. The inactivation of the PTPRJ gene in mice expressing the constitutively active, oncogenic receptor tyrosine kinase FLT3 ITD aggravated known features of leukaemogenesis, revealing PTPRJ's antagonistic role. FLT3 ITD mutations resulting in constitutively kinase activity and cell transformation frequently occur in patients with acute myeloid leukaemia (AML). Thus, in situ activation of PTPRJ could be used to abrogate oncogenic FLT3 signalling. The activity of PTPRJ is suppressed by homodimerization, which is mediated by transmembrane domain (TMD) interactions. Specific Glycine-to-Leucine mutations in the TMD disrupt oligomerization and inhibit the Epidermal Growth Factor Receptor (EGFR) and EGFR-driven cancer cell phenotypes. To study the effects of PTPRJ TMD mutant proteins on FLT3 ITD activity in cell lines, endogenous PTPRJ was inactivated and replaced by stable expression of PTPRJ TMD mutants. Autophosphorylation of wild-type and ITD-mutated FLT3 was diminished in AML cell lines expressing the PTPRJ TMD mutants compared to wild-type-expressing cells. This was accompanied by reduced FLT3-mediated global protein tyrosine phosphorylation and downstream signalling. Further, PTPRJ TMD mutant proteins impaired the proliferation and in vitro transformation of leukemic cells. Although PTPRJ's TMD mutant proteins showed impaired self-association, the specific phosphatase activity of immunoprecipitated proteins remained unchanged. In conclusion, this study demonstrates that the destabilization of PTPRJ TMD-mediated self-association increases the activity of PTPRJ in situ and impairs FLT3 activity and FLT3-driven cell phenotypes of AML cells. Thus, disrupting the oligomerization of PTPRJ in situ could prove a valuable therapeutic strategy to restrict oncogenic FLT3 activity in leukemic cells.

Inhibition of Translation Initiation and Heat Shock Factor 1 (HSF1): A Novel Strategy Targeting FLT3 Double Mutant and Non-FLT3 Mutant AML Progenitor/Stem Cells

The transcription factor Heat shock factor 1 (HSF1) is a key sensor of proteotoxic stress and plays a major role in cancer biology. In various tumors, downregulation of HSF1 inhibits growth and induces cell death, and its activation was reported to be an adverse prognostic factor in breast and lung cancers. We have reported that inhibition of translation represses DNA-binding of HSF1 in cancers; large scale drug screening identified the eIF4a antagonist rohinitib (RHT) as a potent inhibitor of the HSF1 activation state (Santagata et al, Science 2013). Review of public databases provided us with additional rationale for targeting HSF1 and eIF4a in acute myeloid leukemia (AML): 1) mRNA levels of HSPA8, a primary HSF1 target, are correlated with poor prognosis in AML; 2) eIF4a mRNA levels are highest in AML among 12 cancer types; and 3) Gene set enrichment analysis using microarray dataset of functionally-defined leukemia stem cells (LSCs) and non-LSCs (Nature, Ng et al), based on intra-patient comparisons, reveals that a gene set for translation initiation is highly enriched in LSCs (NES 2.73, FDR = 0.000, p= 0.000).RHT induced marked apoptogenic effects in seven human AML cell lines in culture at low nanomolar concentrations (LD50s; 9.5 to 99.5 nM, IC50s; 4.7 to 8.8 nM, based on AnnexinV/PI-positivity at 72hr). RHT inhibited HSF1 transcriptional activity, as shown by 70% reduction in HSPA8 mRNA levels, and 50% reduction in a luciferase reporter driven by the HSPA1A or HSPA6 promoters. We then tested primary samples from 17 AML patients and bone marrow (BM) samples from eight healthy donors. RHT potently induced apoptosis in AML cells, while relatively sparing normal BM cells (%apoptosis: 65.4% vs 6.67%, P < 0.0001). Importantly, a similarly significant difference in sensitivity was also observed between AML and normal stem/progenitor cell populations (CD45+CD34+CD38-). We further found that the activity of RHT was significantly higher in FLT3-ITD than in FLT3-wt AMLs. Comparing isogenic versions of the cell line Ba/F3, RHT more potently killed FLT3-ITD cells than wild-type (wt) cells (LD50s; 65.3 vs 20.1 nM). Immunoblot analysis showed higher activation-associated phospho-HSF1 (Serine 326) in FLT3-ITD Ba/F3 than in FLT3-wt cells, and growth inhibition by HSF1 knockdown was FLT3-ITD-dependent in those lines, indicating greater dependence of FLT3-ITD cells on HSF1. In an AML xenograft model with GFP-luciferase labeled MOLM-13 cells, RHT significantly reduced tumor burden as measured by bioluminescence imaging and reduction in GFP+ leukemic cells peripheral blood (PB) and BM by day 16. Median survival of the treatment group was significantly longer (24 vs 18 days, p < 0.0001).We also found that RHT was synergistic when combined with FLT3 inhibitors of Type I (sorafenib, quizartinib, midostaurin) or Type II (gilteritinib, and E-6201). Immunoblot analysis showed that oncogenic FLT3 activation is associated with increased phosphorylation of HSF1 (S326), suggesting that decreased phosphorylation of the S326 site associated with FLT3 inhibition and diminished DNA-binding by HSF1 driven by eIF4a inhibition combine to promote synergistic lethality. In support of this, HSF1 knockdown sensitized MOLM-13 and FLT3-ITD+ Ba/F3 cells to FLT3 inhibitors. Importantly, similar combinatorial synergistic effects with type II FLT3 inhibitors were observed in cells with dual mutations of FLT3-ITD and FLT3-D835Y/H. This observation has significant clinical relevance because the development of secondary point mutations is a major resistance mechanism against FLT3 inhibitors.Finally, to test the hypothesis that eIF4a inhibition impairs LSC functions, we treated primary patient-derived xenograft AML PDX cells with RHT ex vivo, then injected identical numbers of live cells into recipient NSG mice. Subsequent leukemia engraftment was delayed by one week vs. controls, and human CD45+ cells in PB and BM at 4 weeks post transplantation were significantly reduced (n = 9, 3.30% vs 0.26% in PB, P < 0.0001). This reduced engraftment capacity of RHT-treated PDX AML-LSCs indicates a toxic effect of eIF4a inhibition on AML-LSCs.Collectively, these preclinical findings strongly support the further development of eIF4a inhibitors for the treatment of AML, especially for high-risk FLT3-mutated AML, with additional potential as LSC-targeted therapy. DisclosuresNo relevant conflicts of interest to declare.

Aberrant Bone Homeostasis in AML Is Associated with Activated Oncogenic FLT3-Dependent Cytokine Networks.

Acute myeloid leukaemia (AML) is a haematopoietic malignancy caused by a combination of genetic and epigenetic lesions. Activation of the oncoprotein FLT3 ITD (Fms-like tyrosine kinase with internal tandem duplications) represents a key driver mutation in 25–30% of AML patients. FLT3 is a class III receptor tyrosine kinase, which plays a role in cell survival, proliferation, and differentiation of haematopoietic progenitors of lymphoid and myeloid lineages. Mutant FLT3 ITD results in an altered signalling quality, which causes cell transformation. Recent evidence indicates an effect of FLT3 ITD on bone homeostasis in addition to haematological aberrations. Using gene expression data repositories of FLT3 ITD-positive AML patients, we identified activated cytokine networks that affect the formation of the haematopoietic niche by controlling osteoclastogenesis and osteoblast functions. In addition, aberrant oncogenic FLT3 signalling of osteogenesis-specific cytokines affects survival of AML patients and may be used for prognosis. Thus, these data highlight the intimate crosstalk between leukaemic and osteogenic cells within the osteohaematopoietic niche.

Natural and Semisynthetic Chalcones as Dual FLT3 and Microtubule Polymerization Inhibitors.

Activating mutations in FLT3 receptor tyrosine kinase are found in a third of acute myeloid leukemia (AML) patients and are associated with disease relapse and a poor prognosis. The majority of these mutations are internal tandem duplications (ITDs) in the juxtamembrane domain of FLT3, which have been validated as a therapeutic target. The clinical success of selective inhibitors targeting oncogenic FLT3, however, has been limited due to the acquisition of drug resistance. Herein the identification of a dual FLT3/microtubule polymerization inhibitor, chalcone 4 (2'-allyloxy-4,4'-dimethoxychalcone), is reported through screening of 15 related chalcones for differential antiproliferative activity in leukemia cell lines dependent on FLT3-ITD (MV-4-11) or BCR-ABL (K562) oncogenes and by subsequent screening for mitotic inducers in the HCT116 cell line. Three natural chalcones (1-3) were found to be differentially more potent toward the MV-4-11 (FLT3-ITD) cell line compared to the K562 (BCR-ABL) cell line. Notably, the new semisynthetic chalcone 4, which is a 2'-O-allyl analogue of the natural chalcone 3, was found to be more potent toward the FLT3-ITD+ cell line and inhibited FLT3 signaling in FLT3-dependent cells. An in vitro kinase assay confirmed that chalcone 4 directly inhibited FLT3. Moreover, chalcone 4 induced mitotic arrest in these cells and inhibited tubulin polymerization in both cellular and biochemical assays. Treatment of MV-4-11 cells with this inhibitor for 24 and 48 h resulted in apoptotic cell death. Finally, chalcone 4 was able to overcome TKD mutation-mediated acquired resistance to FLT3 inhibitors in a MOLM-13 cell line expressing FLT3-ITD with the D835Y mutation. Chalcone 4 is, therefore, a promising lead for the discovery of dual-target FLT3 inhibitors.

SRC-Family Kinases in Acute Myeloid Leukaemia and Mastocytosis.

Protein tyrosine kinases have been recognized as important actors of cell transformation and cancer progression, since their discovery as products of viral oncogenes. SRC-family kinases (SFKs) play crucial roles in normal hematopoiesis. Not surprisingly, they are hyperactivated and are essential for membrane receptor downstream signaling in hematological malignancies such as acute myeloid leukemia (AML) and mastocytosis. The precise roles of SFKs are difficult to delineate due to the number of substrates, the functional redundancy among members, and the use of tools that are not selective. Yet, a large num ber of studies have accumulated evidence to support that SFKs are rational therapeutic targets in AML and mastocytosis. These two pathologies are regulated by two related receptor tyrosine kinases, which are well known in the field of hematology: FLT3 and KIT. FLT3 is one of the most frequently mutated genes in AML, while KIT oncogenic mutations occur in 80–90% of mastocytosis. Studies on oncogenic FLT3 and KIT signaling have shed light on specific roles for members of the SFK family. This review highlights the central roles of SFKs in AML and mastocytosis, and their interconnection with FLT3 and KIT oncoproteins.

Wild-type FLT3 and FLT3 ITD exhibit similar ligand-induced internalization characteristics.

Class III receptor tyrosine kinases control the development of hematopoietic stem cells. Constitutive activation of FLT3 by internal tandem duplications (ITD) in the juxtamembrane domain has been causally linked to acute myeloid leukaemia. Oncogenic FLT3 ITD is partially retained in compartments of the biosynthetic route and aberrantly activates STAT5, thereby promoting cellular transformation. The pool of FLT3 ITD molecules in the plasma membrane efficiently activates RAS and AKT, which is likewise essential for cell transformation. Little is known about features and mechanisms of FLT3 ligand (FL)‐dependent internalization of surface‐bound FLT3 or FLT3 ITD. We have addressed this issue by internalization experiments using human RS4‐11 and MV4‐11 cells with endogenous wild‐type FLT3 or FLT3 ITD expression, respectively, and surface biotinylation. Further, FLT3 wild‐type, or FLT3 ITD‐GFP hybrid proteins were stably expressed and characterized in 32D cells, and internalization and stability were assessed by flow cytometry, imaging flow cytometry, and immunoblotting. FL‐stimulated surface‐exposed FLT3 WT or FLT3 ITD protein showed similar endocytosis and degradation characteristics. Kinase inactivation by mutation or FLT3 inhibitor treatment strongly promoted FLT3 ITD surface localization, and attenuated but did not abrogate FL‐induced internalization. Experiments with the dynamin inhibitor dynasore suggest that active FLT3 as well as FLT3 ITD is largely endocytosed via clathrin‐dependent endocytosis. Internalization of kinase‐inactivated molecules occurred through a different yet unidentified mechanism. Our data demonstrate that FLT3 WT and constitutively active FLT3 ITD receptor follow, despite very different biogenesis kinetics, similar internalization and degradation routes.

Characterization of Bypassing Kinases Inferred By Phosphoproteomics in a Midostaurin Resistant Myeloid Cell Line Model

Background: Chronic myeloid neoplasms are heterogeneous malignancies caused by sequential accumulation of genetic lesions in hematopoietic stem cells with a tendency to evolve towards acute myeloid leukemia. Genomic approaches can stratify patients according to their mutational landscape but are limited in predicting response to therapeutic agents. Reliable biomarkers to identify patients' chances for response to targeting compounds remain a crucial necessity in the current post-genomic era of precision medicine. We hypothesize that differential phosphoproteomic (PP) profiles might represent a suitable functional biological layer that allows to identify relevant determinants of oncogenic phenotypes. Aim: The aim of the project was to build a bioinformatics pipeline using PP data to i) identify differentially phosphorylated sites, ii) infer targetable kinases and iii) characterize involved oncogenic pathways. Here, we present results from further exploratory experiments of a previously established PP analysis pipeline that enables to infer sensitive/bypassing kinase activities in a Midostaurin/PKC412 (MIDO) resistant myeloid cell line model. Methods: For the validation of our analysis pipeline, we have previously used the human myeloid cell lines K562 and MOLM13, driven by the oncogenic BCR-ABL1 and FLT3 kinases, and exposed to the kinase inhibitors Nilotinib (NILO) and MIDO, respectively. For the current biological study, we applied our pipeline to explore MIDO resistance mechanisms in published MIDO-resistant (rMOLM13) and sensitive (sMOLM13) cell lines (kindly provided by E. Weisberg, Dana Farber Cancer Institute, USA). r/sMOLM13 were cultured in triplicates for 24hrs and subsequently exposed for 1h to 25nM MIDO or DMSO (CTRL). PPs were enriched with titanium-dioxide and analyzed by mass spectrometry (nanoLC-MS2). Our previously validated Kinase Activity Enrichment Analysis (KAEA) bioinformatics pipeline was further applied to infer kinase activities based on the SetRank enrichment algorithm. KAEA integrates substrate-kinase datasets from five experimentally validated databases complemented with NetworKIN in-silico predictions. The pipeline is supported by a Shiny web-app interface to allow interactive visualization and interrogation of the data. Results: In our previous validation experiments, K562 treated with NILO showed expected inhibition of ABL1 and KIT, whereas MOLM13 treated with MIDO showed expected inhibition of PRKC and downstream kinases of FLT3 (AKT1 and MAPK). In the current biological exploration, we compared rMOLM13 vs sMOLM13 exposed to MIDO and CTRL. rMOLM13 showed slower proliferation and vacuolization compared to sMOLM13. 13,682 phosphorylation sites were identified and the three replicates clustered adequately in the corresponding four conditions sMOLM13_CTRL, sMOLM13_MIDO, rMOLM13_CTRL and rMOLM13_MIDO (fig. 1A). Clusters of over- or underexpressed sites associated with distinct conditions emerged in the heatmap. We performed a KAEA comparison of rMOLM13_MIDO vs sMOLM13_MIDO (fig. 1B) and rMOLM13_CTRL vs sMOLM13_CTRL (fig. 1C). CDK1 and other CDKs enriched as underactive in rMOLM13, in line with the observed reduced proliferation. Most interestingly, kinases PDHK1, PDHK2 and PKM, all pyruvate kinases involved in regulation of mitochondrial metabolism, enriched as overactive, as well as MAPK1/3 and CK2. Reduced proliferation, vacuolization and patterns of enriched kinases pointed towards autophagy as potential resistance mechanism in rMOLM13. We performed preliminary experiments with autophagy inhibitors in combination with MIDO, which supported a synergistic activity (fig. 1D). More results of combined inhibition of the identified kinases will be shown at the meeting. Conclusions: With our PP analysis pipeline, we were able to characterize, in unanticipated detail, dynamic changes of PP profiles, kinase activities and potentially involved oncogenic pathways in a MIDO resistant myeloid cell line model. We identified biologically relevant mechanisms of resistance that will be explored as potential targets for combination therapy. Our experiments in myeloid cell lines provide a proof of concept for our analysis pipeline. They allow us to move forward to the analysis of primary patient samples and promote precision medicine based on functional biomarkers identified by PP in patients with myeloid neoplasms. Disclosures Jankovic: Celgene: Other: financial support for travel. Bonadies:Roche: Other: financial support for travel, Research Funding; Novartis: Other: financial support for travel, Research Funding; Celgene: Other: financial support for travel, Research Funding; Janssen: Other: financial support for travel; Amgen: Other: financial support for travel; Sanofi Genzyme: Other: financial support for travel.

Abstract 27: Identification and characterization of a novel dual FLT3/microtubule polymerization inhibitor through cell-based screening

Abstract Activating mutations in FLT3 receptor tyrosine kinase are found in one-third of acute myeloid leukemia (AML) patients and are associated with disease relapse and bad prognosis. Majority of these mutations are Internal tandem duplications (ITD) in the juxtamembrane domain of FLT3 and have been validated as a therapeutic target. Clinical success of selective inhibitors targeting oncogenic FLT3, however, has been limited due to the acquisition of drug resistance. Here we report the identification of a dual FLT3/microtubule polymerization inhibitor, HH-IA-208, through cell-based screenings for the differential killing of leukemia cell lines expressing FLT3-ITD (MV411) or BCR-ABL (K562) using an MTS proliferation assay and for mitotic inducers using a dot-blot assay for histone H3 phosphorylation in HCT116 cell line. HH-IA-208, a chalcone derivative, inhibits FLT3 in vitro and treatment of FLT3-ITD+ cell lines with HH-IA-208 results in reduced FLT3 signaling. Similarly, HH-IA-208 inhibits tubulin polymerization in a biochemical assay and in cells that results in a mitotic arrest. HH-IA-208 is selectively more potent in leukemia cell lines expressing FLT3-ITD and treatment for 24 and 48 hours with the inhibitor results in apoptotic cell death. Furthermore, HH-IA-208 is able to overcome TKD mutation-mediated acquired resistance to FLT3 inhibitors in a MOLM13 cell line expressing FLT3-ITD with the D835Y mutation. HH-IA-208, therefore, is a promising lead for the discovery of dual target FLT3 inhibitors that can overcome resistance to selective FLT3 inhibitors. Citation Format: Haleema Sadia Malik, Aishah Bilal, Rahim Ullah, Maheen Iqbal, Rahman Shah Saleem, Hidayat Hussain, Amir Faisal. Identification and characterization of a novel dual FLT3/microtubule polymerization inhibitor through cell-based screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 27.

PF281 CLI24–001: FIRST IN HUMAN STUDY OF SEL24/MEN1703, AN ORAL DUAL PIM/FLT3 KINASE INHIBITOR, IN PATIENTS WITH ACUTE MYELOID LEUKEMIA

Background:PIM kinases are oncogenic FLT3 targets expressed in acute myeloid leukemia (AML) cells. Increased PIM expression is found in samples of relapsed AML patients exposed to FLT3 inhibitors (FLT3i). There is preclinical evidence that PIM inhibition restores cell sensitivity to FLT3i, and that dual FLT3/PIM inhibition eradicates FLT3‐ITD+ primary AML cells. SEL24/MEN1703, a potent PIM/FLT3 dual inhibitor, showed a significantly broader spectrum of activity in AML cell lines and primary AML blasts, irrespective of FLT3 status compared to FLT3 or PIM inhibitors aloneAims:To determine the recommended phase 2 dose (RP2D), the PK profile and the single agent activity of SEL24/MEN1703 in relapsed/refractory or newly diagnosed AML patients (excluding APL) unsuitable to chemotherapyMethods:First in Human, open label, non‐randomized, multi‐center, dose‐escalation (DE) and cohort expansion (CE) study of oral SEL24/MEN1703. SEL24/MEN1703 is given orally, QD, FOR 14 days in a 21‐day cycle with cycles repeated until disease progression or unacceptable toxicity. Dose escalation follows a 3+3 design to identify the RP2D; CE at RP2D is planned to confirm the safety profile and assess single agent activity. Patients are eligible for the study regardless of mutations and/or prior FLT3i treatment; prior PIM inhibitors are not allowed. White blood count (WBC) of ≤30 x 109/L is required at baseline. Key secondary objectives include PK profile and single agent anti‐leukemic activity; exploratory objectives include assessment of clinical activity in relevant AML subsets of patients (eg FLT3 mutated)Results:As of January 8th, 2019 (data cutoff), n = 17 patients were treated and the study is enrolling. In the RP2D expansion phase, the study (currently running in 5 US sites) will be expanded to approximately 40 sites in the US and EUSummary/Conclusion:SEL24/MEN1703, a first in class, oral dual PIM/FLT3‐ITD inhibitor, is completing the DE part of a two‐part FIH study in patients with relapsed/refractory or unfit newly diagnosed AML unsuitable to chemotherapy. The RP2D expansion phase is planned to involve either US and EU investigational sites. This is the first clinical trial testing a dual PIM/FLT3‐ITD inhibitor, potentially active in AML regardless of FLT3 status and able to overcome FLT3i resistance

Glucocorticoid-resistant B cell acute lymphoblastic leukemia displays receptor tyrosine kinase activation

The response of childhood acute lymphoblastic leukemia (ALL) to dexamethasone predicts the long-term remission outcome. To explore the mechanisms of dexamethasone resistance in B cell ALL (B-ALL), we generated dexamethasone-resistant clones by prolonged treatment with dexamethasone. Using RNA-sequencing and high-throughput screening, we found that dexamethasone-resistant cells are dependent on receptor tyrosine kinases. Further analysis with phosphokinase arrays showed that the type III receptor tyrosine kinase FLT3 is constitutively active in resistant cells. Targeted next-generation and Sanger sequencing identified an internal tandem duplication mutation and a point mutation (R845G) in FLT3 in dexamethasone-resistant cells, which were not present in the corresponding sensitive clones. Finally, we showed that resistant cells displayed sensitivity to second-generation FLT3 inhibitors both in vitro and in vivo. Collectively, our data suggest that long-term dexamethasone treatment selects cells with a distinct genetic background, in this case oncogenic FLT3, and therefore therapies targeting FLT3 might be useful for the treatment of relapsed B-ALL patients.

Abstract A080: A phase 1 study of single-agent pexidartinib in Asian patients with advanced solid tumors (NCT02734433)

Abstract Background: Pexidartinib is a novel, orally active, small-molecule kinase inhibitor that selectively targets the colony-stimulating factor-1 receptor (CSF1R), as well as the receptors c-kit and oncogenic Flt3. Based on these targets, pexidartinib may inhibit tumor growth directly by blocking oncogenic drivers such as CSF-1, c-kit, and Flt3, or indirectly by modulating the tumor microenvironment and interactions between stromal cells and tumors. This first study in Asia evaluated the safety, tolerability, and pharmacokinetic (PK) profile in an Asian population. Methods: This phase 1 non-randomized, open-label, multiple-dose study evaluated the safety and tolerability of pexidartinib in Asian patients with advanced solid tumors. This dose-escalation study employed a 3+3 design and included 2 dose levels. Patients at dose level 1 received 600 mg/day and patients at dose level 2 received 1000 mg/day for the first 2 weeks followed by 800 mg/day thereafter. Patients still receiving treatment after four 28-day cycles were eligible to continue pexidartinib in an extension phase. Treatment continued until disease progression, unacceptable toxicity, or withdrawal of consent. Study objectives included assessment of pexidartinib safety and tolerability, PK, and pharmacodynamic effects on circulating CSF-1 and adiponectin, as well as determination of the maximum tolerated and recommended phase 2 doses (MTD and RP2D). Results: Eleven patients (6 males and 5 females, median age 64, range 23-82) were treated with pexidartinib in the dose-escalation phase. Preliminary PK data indicate that exposure (Cmax and AUC0-8h) increased proportionally with dose and the plasma concentration level increased after multiple doses. CSF-1 and adiponectin plasma concentrations increased after administration of pexidartinib. Grade ≥3 adverse events (AEs) observed were increased alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma-glutamyl transferase, and bilirubin, as well as anemia and lower back pain. No patients experienced dose-limiting toxicities, and the MTD was determined to be 1000 mg/day. At the time of this analysis, 7 patients had experienced disease progression, 3 patients had withdrawn from the study, and 1 patient with tenosynovial giant cell tumor continued to receive pexidartinib for more than 341 days. Conclusions: Pexidartinib was determined to be safe and tolerable at the 1000 mg/day dose for Asian patients, and the most common treatment-related AEs were elevated liver enzyme levels. Pexidartinib demonstrated clinical activity in a patient with tenosynovial giant cell tumor, and is being evaluated in a phase 3 study for patients with tenosynovial giant cell tumors in Western countries (NCT02371369). Citation Format: Chia-Chi Lin, Jih-Hsiang Lee, Chih-Hung Hsu, Wei-Wu Chen, Yu-Hsin Yen, Chih-Hsin Yang, Ling Zhang, Shun-ichi Sasaki, Lillian Chiu, Ann-Lii Cheng. A phase 1 study of single-agent pexidartinib in Asian patients with advanced solid tumors (NCT02734433) [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A080.

Abstract PR04: Degradation of leukemia oncogenes: A novel approach to therapy of leukemia

Abstract AML is a heterogeneous group of malignancies with significant variability in phenotype and mechanisms of leukemogenesis. Despite our increased understanding of the genetic abnormalities that drive AML, very few targeted therapeutic agents have yet been developed. The prevailing approach to drugging oncoproteins is direct targeting of the mutant protein with a small molecule or antibody. Unfortunately, this strategy has not proven particularly fruitful in the case of AML as many mutant driver proteins are “undruggable” and the targetable enzymes (i.e., oncogenic FLT3) rapidly acquire resistance to inhibitors in the clinic. We are pursuing a novel and innovative approach that focuses on promoting degradation of AML oncoproteins through small-molecule inhibition of the cellular machinery that regulates turnover. Many oncoproteins are tagged for degradation by the proteasome or lysosome by post-translational addition of polyubiquitin chains. The process is, however, highly dynamic and reversible by deubiquitylating enzymes (DUBs), which cleave ubiquitin from substrate proteins. A subset of AML mutant drivers, including RAS, FLT3, and PML-RARα, have been found to undergo ubiquitin-mediated degradation; however, the DUB enzymes stabilizing them are unknown. In order to identify novel targets and compounds that regulate protein homeostasis of leukemic oncoproteins, we employed whole-cell phenotypic screens of most reported small-molecule DUB inhibitors, annotated for inhibitory activity across a broad panel of DUBS, using oncogene-dependent and control cell lines followed by hit validation and target deconvolution. Using this approach, we identified inhibitors, and candidate DUBs, involved in regulation of protein levels of mutant FLT3, mutant JAK2, mutant KRAS, and PML-RARα;. Follow-up work that focused on FLT3-ITD identified USP10 as a DUB that stabilizes the oncoprotein via removal of a degradative ubiquitin tag. Furthermore, we show that pharmacologic inhibition of USP10 promotes degradation of FLT3-ITD but not wild-type (wt) FLT3, leads to selective killing of oncogenic FLT3-expressing AML cells in vitro and in vivo, and overrides resistance to FLT3 kinase inhibitors caused by tyrosine kinase domain (TKD) mutations and other mechanisms. Our studies hence validate USP10 as a therapeutic target for FLT3 mutant-positive AML. Building on this work, we executed a medicinal chemistry optimization effort that has yielded USP10 inhibitors with improved potency, and selectivity and mechanism studies have yielded insights into the origin of specificity for mutant versus wt FLT3. Furthermore, we have validated multiple DUB inhibitors that promote selective degradation of V617F JAK2 and intriguingly, identified compounds efficacious, in terms of inhibition of proliferation, across multiple genetic backgrounds. To the best of our knowledge, this is the first identification of a novel DUB substrate using a DUB-targeting small-molecule library screen and the first demonstration of stabilization of a mutant driver oncoprotein in AML by a DUB enzyme. DUB inhibitors have the potential to eliminate oncoproteins, but it is early yet in the field. The first DUB inhibitor clinical trial is starting this year for multiple myeloma. Our work establishing the therapeutic potential of DUB inhibitors for oncogenes including PML-RARα; and constitutively activated FLT3, JAK2, and RAS, as well as potentially other oncoprotein-driven AML patient populations, will lay the groundwork for development of first-in-class drugs for AML. This abstract is also being presented as Poster 10. Citation Format: Sara Buhrlage, Ellen Weisberg, Ilaria Lamberto, Jing Yang, Nathan Schauer, Martin Sattler, Atushi Nonami, Amanda Christie, David Weinstock, Stella Ritorto, Virginia DeCesare, Matthias Trost, Richard Stone, Nathanael Gray, James Griffin. Degradation of leukemia oncogenes: A novel approach to therapy of leukemia [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr PR04.

Spotlight on midostaurin in the treatment of FLT3-mutated acute myeloid leukemia and systemic mastocytosis: design, development, and potential place in therapy.

The Fms-like tyrosine kinase-3 (FLT3; fetal liver kinase-2; human stem cell tyrosine kinase-1; CD135) is a class III receptor tyrosine kinase that is normally involved in regulating the proliferation, differentiation, and survival of both hematopoietic cells and dendritic cells. Mutations leading it to be constitutively activated make it an oncogenic driver in ~30% of acute myeloid leukemia (AML) patients where it is associated with poor prognosis. The prevalence of oncogenic FLT3 and the dependency on its constitutively activated kinase activity for leukemia growth make this protein an attractive target for therapeutic intervention. Of the numerous small molecule inhibitors under clinical investigation for the treatment of oncogenic FLT3-positive AML, the N-benzoyl-staurosporine, midostaurin (CGP41251; PKC412; Rydapt®; Novartis Pharma AG, Basel, Switzerland), is the first to be approved by the US Food and Drug Administration for the treatment, in combination with standard chemotherapy, of newly diagnosed adult AML patients who harbor mutations in FLT3. Here, we describe the early design of midostaurin, the preclinical discovery of its activity against oncogenic FLT3, and its subsequent clinical development as a therapeutic agent for FLT3 mutant-positive AML.

Inhibition of USP10 induces degradation of oncogenic FLT3.

Oncogenic forms of the kinase FLT3 are important therapeutic targets in acute myeloid leukemia (AML); however, clinical responses to small-molecule kinase inhibitors are short-lived as a result of the rapid emergence of resistance due to point mutations or compensatory increases in FLT3 expression. We sought to develop a complementary pharmacological approach whereby proteasome-mediated FLT3 degradation could be promoted by inhibitors of the deubiquitinating enzymes (DUBs) responsible for cleaving ubiquitin from FLT3. Because the relevant DUBs for FLT3 are not known, we assembled a focused library of most reported small-molecule DUB inhibitors and carried out a cellular phenotypic screen to identify compounds that could induce the degradation of oncogenic FLT3. Subsequent target deconvolution efforts allowed us to identify USP10 as the critical DUB required to stabilize FLT3. Targeting of USP10 showed efficacy in preclinical models of mutant-FLT3 AML, including cell lines, primary patient specimens and mouse models of oncogenic-FLT3-driven leukemia.

Characterization of midostaurin as a dual inhibitor of FLT3 and SYK and potentiation of FLT3 inhibition against FLT3-ITD-driven leukemia harboring activated SYK kinase.

Oncogenic FLT3 kinase is a clinically validated target in acute myeloid leukemia (AML), and both multi-targeted and selective FLT3 inhibitors have been developed. Spleen tyrosine kinase (SYK) has been shown to be activated and increased in FLT3-ITD-positive AML patients, and has further been shown to be critical for transformation and maintenance of the leukemic clone in these patients. Further, over-expression of constitutively activated SYK causes resistance to highly selective FLT3 tyrosine kinase inhibitors (TKI). Up to now, the activity of the multi-targeted FLT3 inhibitor, midostaurin, against cells expressing activated SYK has not been explored in the context of leukemia, although SYK has been identified as a target of midostaurin in systemic mastocytosis. We compared the ability of midostaurin to inhibit activated SYK in mutant FLT3-positive AML cells with that of inhibitors displaying dual SYK/FLT3 inhibition, targeted SYK inhibition, and targeted FLT3 inhibition. Our findings suggest that dual FLT3/SYK inhibitors and FLT3-targeted drugs potently kill oncogenic FLT3-transformed cells, while SYK-targeted small molecule inhibition displays minimal activity. However, midostaurin and other dual FLT3/SYK inhibitors display superior anti-proliferative activity when compared to targeted FLT3 inhibitors, such as crenolanib and quizartinib, against cells co-expressing FLT3-ITD and constitutively activated SYK-TEL. Interestingly, additional SYK suppression potentiated the effects of dual FLT3/SYK inhibitors and targeted FLT3 inhibitors against FLT3-ITD-driven leukemia, both in the absence and presence of activated SYK. Taken together, our findings have important implications for the design of drug combination studies in mutant FLT3-positive patients and for the design of future generations of FLT3 inhibitors.

Abstract 2371: Oncogenic transformation of FLT3-ITD is partially dependent on tyrosine 842 in the activation loop

Abstract Acute myeloid leukemia (AML) is a heterogeneous disease of the blood. About 30 % of AML patients carry an oncogenic mutant of the type III receptor tyrosine kinase FLT3. Among the various mutations in FLT3, the internal tandem duplication (ITD) mutations in the juxtamembrane domain are the most common type of mutation, while point mutations in the kinase domain also occur. Although oncogenic FLT3 mutants are constitutively active, the wild-type receptor needs its ligand (FLT3 ligand, FL) for activation. Ligand binding to the receptor induces dimerization, auto-phosphorylation, and activation of the receptor. Tyrosine phosphorylation sites on FLT3 constitute docking sites for interacting proteins that transduce pro-survival and proliferative signals. The so-called activation loop tyrosine residue (in FLT3, Y842) is well conserved in all receptor tyrosine kinases and long been known essential for the activity of some but not all receptor tyrosine kinases. Recently we have shown that activation loop tyrosine residue in KIT is not essential for its activation but plays an important role in receptor stabilization and downstream signaling. However, so far the role of FLT3 activation loop tyrosine residue remains unknown. We generated 32D cell lines carrying a Y-to-F mutant (Y842F) of FLT3 in either the wild-type or ITD background to study the role of this tyrosine residue. We show that the Y842 residue is not necessary for FLT3 activation or stability but is required for oncogenic transformation. We observed that Y842F mutation results in reduced cell viability, proliferation and in vitro colony formation in semi-solid medium. In addition, cells expressing the Y842F mutant in FLT3-ITD background display a significant reduction in tumor volume and weight in a xenograft model in nude mice. Gene set enrichment analysis (GESA) shows that mutation of Y842 causes suppression of anti-apoptotic genes. Furthermore, we demonstrated that phosphorylated Y842 is one of the binding sites of the protein tyrosine phosphataseSHP2, which is required for activation of signaling through the RAS/ERK pathway. Taken together, our data suggest that activation loop tyrosine residue in FLT3 plays an important role in the cellular transformation which is regulated by RAS/ERK signaling. Citation Format: Julhash U. Kazi, Lars Rönnstrand. Oncogenic transformation of FLT3-ITD is partially dependent on tyrosine 842 in the activation loop [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 2371. doi:10.1158/1538-7445.AM2017-2371

Acute myeloid leukemia cells require 6-phosphogluconate dehydrogenase for cell growth and NADPH-dependent metabolic reprogramming.

Acute myeloid leukemia (AML) cells are highly dependent on glycolytic pathways to generate metabolic energy and support cell growth, hinting at specific, targetable vulnerabilities as potential novel targets for drug development. Elevated levels of NADPH, a central metabolic factor involved in redox reactions, are common in myeloid leukemia cells, but the significance or biochemical basis underlying this increase is unknown. Using a small molecule analog that efficiently inhibits NADPH-producing enzymes, we found that AML cells require NADPH homeostasis for cell growth. We also found that inhibiting NADPH production through knockdown of 6-phosphogluconate dehydrogenase (6PGD) within the pentose phosphate pathway was sufficient to reduce cell growth and lactate production, a measure of metabolic reprogramming. Further, inhibition of 6PGD activity reduced NADH levels and enzymatic activity of the oxidized NADH-dependent sirtuin-1. Targeting 6PGD and NADPH production was sufficient to block growth of AML cell lines resistant to the chemotherapeutics daunorubicin and cytarabine. Importantly, stromal cell-mediated resistance to targeted inhibition of oncogenic FLT3 kinase activity by quizartinib was circumvented by 6PGD knockdown. Overall, these data suggest that the dependency of AML cells on NADPH to permit increased glycolytic flux creates a potential vulnerability of possible therapeutic benefit, since much of the enhanced production of NADPH is dependent on the activity of a single enzyme, 6PGD.

Tyrosine 842 in the activation loop is required for full transformation by the oncogenic mutant FLT3-ITD

The type III receptor tyrosine kinase FLT3 is frequently mutated in acute myeloid leukemia. Oncogenic FLT3 mutants display constitutive activity leading to aberrant cell proliferation and survival. Phosphorylation on several critical tyrosine residues is known to be essential for FLT3 signaling. Among these tyrosine residues, Y842 is located in the so-called activation loop. The position of this tyrosine residue is well conserved in all receptor tyrosine kinases. It has been reported that phosphorylation of the activation loop tyrosine is critical for catalytic activity for some but not all receptor tyrosine kinases. The role of Y842 residue in FLT3 signaling has not yet been studied. In this report, we show that Y842 is not important for FLT3 activation or ubiquitination but plays a critical role in regulating signaling downstream of the receptor as well as controlling receptor stability. We found that mutation of Y842 in the FLT3-ITD oncogenic mutant background reduced cell viability and increased apoptosis. Furthermore, the introduction of the Y842 mutation in the FLT3-ITD background led to a dramatic reduction in in vitro colony forming capacity. Additionally, mice injected with cells expressing FLT3-ITD/Y842F displayed a significant delay in tumor formation, compared to FLT3-ITD expressing cells. Microarray analysis comparing gene expression regulated by FLT3-ITD versus FLT3-ITD/Y842F demonstrated that mutation of Y842 causes suppression of anti-apoptotic genes. Furthermore, we showed that cells expressing FLT3-ITD/Y842F display impaired activity of the RAS/ERK pathway due to reduced interaction between FLT3 and SHP2 leading to reduced SHP2 activation. Thus, we suggest that Y842 is critical for FLT3-mediated RAS/ERK signaling and cellular transformation.

MicroRNA-194 Promotes Prostate Cancer Metastasis by Inhibiting SOCS2.

Serum levels of miR-194 have been reported to predict prostate cancer recurrence after surgery, but its functional contributions to this disease have not been studied. Herein, it is demonstrated that miR-194 is a driver of prostate cancer metastasis. Prostate tissue levels of miR-194 were associated with disease aggressiveness and poor outcome. Ectopic delivery of miR-194 stimulated migration, invasion, and epithelial-mesenchymal transition in human prostate cancer cell lines, and stable overexpression of miR-194 enhanced metastasis of intravenous and intraprostatic tumor xenografts. Conversely, inhibition of miR-194 activity suppressed the invasive capacity of prostate cancer cell lines in vitro and in vivo Mechanistic investigations identified the ubiquitin ligase suppressor of cytokine signaling 2 (SOCS2) as a direct, biologically relevant target of miR-194 in prostate cancer. Low levels of SOCS2 correlated strongly with disease recurrence and metastasis in clinical specimens. SOCS2 downregulation recapitulated miR-194-driven metastatic phenotypes, whereas overexpression of a nontargetable SOCS2 reduced miR-194-stimulated invasion. Targeting of SOCS2 by miR-194 resulted in derepression of the oncogenic kinases FLT3 and JAK2, leading to enhanced ERK and STAT3 signaling. Pharmacologic inhibition of ERK and JAK/STAT pathways reversed miR-194-driven phenotypes. The GATA2 transcription factor was identified as an upstream regulator of miR-194, consistent with a strong concordance between GATA2 and miR-194 levels in clinical specimens. Overall, these results offer new insights into the molecular mechanisms of metastatic progression in prostate cancer. Cancer Res; 77(4); 1021-34. ©2016 AACR.

The GLI-Code As a Potential Therapeutic Target in Myeloid Neoplasms

The proliferation of hematopoietic stem cells (HSCs) and progenitor cells (HPC) is tightly regulated during physiologic function of the hematopoietic system. Recent data suggest a role for activated developmental signaling pathways such as Hedgehog (Hh), Notch and Wnt in this process. The GLI genes (GLI1-4), known as GLI-code, encode Cys2-His2 zinc finger transcription factors that regulate the expression of target genes at the distal end of the canonical Hh pathway. This pathway is important during embryogenesis but mostly inactive in normal adult tissues. Activation of Hh effector SMO triggers an intracellular signaling cascade leading to activation of GLI2 and GLI3, which ultimately activate GLI1. It is well established that activation of GLI transcription factors may occur independent of canonical Hh signaling. Aberrant activation of the Hh/GLI pathway has been implicated in tumor progression and resistance to chemotherapy in MDS/AML. GLI transcription factors serve as a common node of activation through which many oncogenic signals, e.g., Hh, FLT3, PI3K-AKT, TGFβ and RAS converge. Moreover, overexpression of GLI transcription factors constitutes a negative prognostic survival marker. Higher expression of GLI transcription factors were recently reported in mutant FLT3-ITD AML cases. Extensive analysis of publicly available gene expression data sets revealed that GLI1 expression was significantly higher in complex AML karyotype (p<0.001, n=48) compared to other cytogenetic abnormalities t(15,17), inv(16), t(8,21), and t(11q23) (n=54, 47, 60 and 43 respectively). GLI1 expression was also significantly higher (p<0.05) in complex AML karyotype (n=87) compared to normal karyotype cases (n=989). The expression of GLI1 was even higher (p<0.001) in MDS (n=228) than normal AML karyotype. In addition, 8 out of 14 of NRAS mutant and 5 out of 7 KRAS mutant cases of AML in TCGA database have higher GLI1 expression. These biomarkers indicate advanced disease and constitute a major clinical problem with limited therapeutic options. Our observations strongly suggest that the GLI1 would be a bona-fide therapeutic target in MDS/AML. Thus specific and potent inhibitors of GLI1 can provide a rational approach for the treatments of these cases.While most efforts have been directed towards therapeutic targeting of upstream Hh signaling through PTCH/SMO, these measures remain ineffective in the presence of parallel signaling converging at GLI independent of canonical Hh pathway. Therefore, there is a need for the development of a GLI1-specific inhibitor that effectively blocks the nodal channel of Hh signaling. For this purpose, we used a structure guided approach to develop a highly potent and specific GLl1/GLI2 inhibitor iGli702, capable of preventing GLI1 DNA binding and thereby induction of cell death. Computational, biochemical and biophysical analysis demonstrated that iGli702 binds to the DNA binding surface of GLI1 protein between Zn-finger 2 and 3, effectively preventing Gli1-DNA interaction. Our data show that iGli702 is over 40 fold more effective than the reported GLI1/2 antagonist GANT61 in AML cell lines. iGli702 promotes apoptosis in a panel of leukemic cell lines, primary patient-derived AML samples and animal models. We observed an IC50 of 150 nM in cell viability assays.To test whether iGli702 induces cell death of progenitor tumor cells in semi-solid methylcellulose-based media, sorted CD34 positive tumor cells from AML cell lines (MOLM-13 and THP1) were treated with iGli702. This treatment significantly reduced colony formation of leukemia cells. However, iGli702 has only a minimal effect on normal PBMC and BM cells (LD50 >10µM). iGli702 efficacy was also compared in vitro to common chemotheraputic agents in AML. CytA and Ida were applied in parallel with iGli702to MOLM-13, THP-1, K562 AML cell lines. iGli702 showed superior and equivalent activity to CytA and Ida, respectively. In addition, we applied deep RNA NGS to analyze the cellular transcriptome after treatment with iGli702. Our data suggest that iGli702 directly inhibits Hedgehog pathway by reducing Hh target genes expression. In summary, iGli702 is highly effective as a single agent in killing hematopoietic malignant cells. This in turn demonstrates that targeting GLI1 Hh effector node is an excellent therapeutic approach in myeloid neoplasms. DisclosuresNo relevant conflicts of interest to declare.