BackgroundActivating mutations of class III receptor tyrosine kinases (RTK) FLT3, PDGFR and KIT are associated with multiple human neoplasms including hematologic malignancies, for example: systemic mast cell disorders (KIT), non-CML myeloproliferative neoplasms (PDGFR) and subsets of acute leukemias (FLT3 and KIT). First generation tyrosine kinase inhibitors (TKI) are rapidly being integrated into routine cancer care. However, the expanding spectrum of TK-mutations, bioavailability issues and the emerging problem of primary or secondary TKI-therapy resistance have lead to the search for novel second generation TKIs to improve target potency and to overcome resistant clones.Quizartinib was recently demonstrated to be a selective FLT3 inhibitor with excellent pharmacokinetics and promising in vivo activity in a phase II study for FLT3 ITD + AML patients. In vitro kinase assays have suggested that in addition to FLT3, quizartinib also targets related class III RTK isoforms.MethodsVarious FLT3 or KIT leukemia cell lines and native blasts were used to determine the antiproliferative and proapoptotic efficacy of quizartinib. To better compare differences between the mutant kinase isoforms, we generated an isogenic BaF3 cell line expressing different FLT3, KIT or BCR/ABL isoforms. Using immunoblotting, we examined the effects of quizartinib on activation of mutant KIT or FLT3 isoforms.ResultsKinase inhibition of (mutant) KIT, PDGFR and FLT3 isoforms by quizartinib leads to potent inhibition of cellular proliferation and induction of apoptosis in in vitro leukemia models as well as in native leukemia blasts treated ex vivo. However, the sensitivity patterns vary widely depending on the underlying (mutant)-kinase isoform, with some isoforms being relatively insensitive to this agent (e.g. FLT3 D835V and KIT codon D816 mutations). Evaluation of sensitivities in an isogenic cellular background confirms a direct association with the underlying mutant-TK isoform – which is further validated by immunoblotting experiments demonstrating kinase inhibition consistent with the cellular sensitivity/resistance to quizartinib.ConclusionQuizartinib is a potent second-generation class III receptor TK-inhibitor – but specific, mutation restricted spectrum of activity may require mutation screening prior to therapy.

BackgroundDysregulation of the PI3Kinase/AKT pathway is involved in the pathogenesis of many human malignancies. In acute leukemia, the AKT pathway is frequently activated, however mutations in the PI3K/AKT pathway are uncommon. In some cases, constitutive AKT activation can be linked to gain-of-function tyrosine kinase (TK) mutations upstream of the PI3K/AKT pathway. Inhibitors of the PI3K/AKT pathway are attractive candidates for cancer drug development, but so far clinical efficacy of PI3K inhibitors against various neoplasms has been moderate. Furthermore, specific MTORC1 inhibitors, acting downstream of AKT, have the disadvantage of activating AKT via feed-back mechanisms. We now evaluated the antitumor efficacy of NVP-BGT226, a novel dual pan-PI3K and MTORC1/2 inhibitor, in acute leukemia.MethodsNative leukemia blasts were stained to analyze for AKT phosphorylation levels on a flow cytometer. Efficacy of NVP-BGT226 in comparison to a second dual inhibitor, NVP-BEZ235, was determined with regard to cellular proliferation, autophagy, cell cycle regulation and induction of apoptosis in in vitro and ex vivo cellular assays as well as on the protein level. An isogenic AKT-autoactivated Ba/F3 model, different human leukemia cell lines as well as native leukemia patient blasts were studied. Isobologram analyses were set up to calculate for (super) additive or antagonistic effects of two agents.ResultsWe show, that phosphorylation of AKT is frequently augmented in acute leukemia. NVP-BGT226 as well as NVP-BEZ235 profoundly and globally suppress AKT signaling pathways, which translates into potent antiproliferative effects. Furthermore, NVP-BGT226 has potent proapoptotic effects in vitro as well as in ex vivo native blasts. Surprisingly and in contrast, NVP-BEZ235 leads to a profound G1/G0 arrest preventing significant induction of apoptosis. Combination with TK inhibitors, which are currently been tested in the treatment of acute leukemia subtypes, overcomes cell cycle arrest and results in (super)additive proapoptotic effects for NVP-BGT226 – but also for NVP-BEZ235. Importantly, mononuclear donor cells show lower phospho-AKT expression levels and consequently, relative insensitivity towards dual PI3K-MTORC1/2 inhibition.ConclusionsOur data suggest a favorable antileukemic profile for NVP-BGT226 compared to NVP-BEZ235 – which provides a strong rationale for clinical evaluation of the dual PI3K-MTORC1/2 inhibitor NVP-BGT226 in acute leukemia.

Background Apoptosis-stimulating Protein of TP53-2 ( ASPP2 ) is a tumor suppressor enhancing TP53-mediated apoptosis via binding to the TP53 core domain. TP53 mutations found in cancers disrupt ASPP2 binding, arguing for an important role of ASPP2 in TP53-mediated tumor suppression. We now identify an oncogenic splicing variant, ASPP2κ , with high prevalence in acute leukemia. Methods An mRNA screen to detect ASPP2 splicing variants was performed and ASPP2κ was validated using isoform-specific PCR approaches. Translation into a genuine protein isoform was evaluated after establishing epitope-specific antibodies. For functional studies cell models with forced expression of ASPP2κ or isoform-specific ASPP2κ -interference were created to evaluate proliferative, apoptotic and oncogenic characteristics of ASPP2κ . Findings Exon skipping generates a premature stop codon, leading to a truncated C-terminus, omitting the TP53-binding sites. ASPP2κ translates into a dominant-negative protein variant impairing TP53-dependent induction of apoptosis. ASPP2κ is expressed in CD34+ leukemic progenitor cells and functional studies argue for a role in early oncogenesis, resulting in perturbed proliferation and impaired induction of apoptosis, mitotic failure and chromosomal instability (CIN) – similar to TP53 mutations. Importantly, as expression of ASPP2κ is stress-inducible it defines a novel class of dynamic oncogenes not represented by genomic mutations. Interpretation Our data demonstrates that ASPP2κ plays a distinctive role as an antiapoptotic regulator of the TP53 checkpoint, rendering cells to a more aggressive phenotype as evidenced by proliferation and apoptosis rates – and ASPP2 κ expression results in acquisition of genomic mutations, a first initiating step in leukemogenesis. We provide proof-of-concept to establish ASPP2κ as a clinically relevant biomarker and a target for molecule-defined therapy. Fund Unrestricted grant support from the Wilhelm Sander Foundation for Cancer Research, the IZKF Program of the Medical Faculty Tübingen, the Brigitte Schlieben-Lange Program and the Margarete von Wrangell Program of the State Ministry Baden-Wuerttemberg for Science, Research and Arts and the Athene Program of the excellence initiative of the Eberhard-Karls University, Tübingen.

BackgroundIt has been previously demonstrated in several cancer models, that Dronabinol (THC) may have anti-tumor activity – however, controversial data exists for acute leukemia. We have anecdotal evidence that THC may have contributed to disease control in a patient with acute undifferentiated leukemia.MethodsTo test this hypothesis, we evaluated the antileukemic efficacy of THC in several leukemia cell lines and native leukemia blasts cultured ex vivo. Expression analysis for the CB1/2 receptors was performed by Western immunoblotting and flow cytometry. CB-receptor antagonists as well as a CRISPR double nickase knockdown approach were used to evaluate for receptor specificity of the observed proapoptotic effects.ResultsMeaningful antiproliferative as well as proapoptotic effects were demonstrated in a subset of cases – with a preference of leukemia cells from the lymphatic lineage or acute myeloid leukemia cells expressing lymphatic markers. Induction of apoptosis was mediated via CB1 as well as CB2, and expression of CB receptors was a prerequisite for therapy response in our models. Importantly, we demonstrate that antileukemic concentrations are achievable in vivo.ConclusionOur study provides rigorous data to support clinical evaluation of THC as a low-toxic therapy option in a well defined subset of acute leukemia patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-2029-8) contains supplementary material, which is available to authorized users.

tion as a novel (drugable) field for future leukemia research. Funding: Unrestricted grant support by the IZKF Program of the Medical Faculty T€ ubingen (MMS) and Brigitte Schlieben-Lange Program as well as the Margarete von Wrangell Program of the Ministry of Science, Research and the Arts, Baden-W€ urttemberg, Germany (KKS) and Athene Program of the excellence initiative University of T€ ubingen (KKS).

Activating mutations of the class III receptor tyrosine kinases FLT3 and KIT are associated with certain human neoplasms, including hematologic malignancies, i.e. the majority of patients with systemic mast cell disorders (KIT) and subsets of patients with acute myelogenous leukemia (FLT3 and KIT). Crenolanib is a potent selective FLT3 inhibitor with high efficacy against internal tandem dupliction mutations (ITD) – but also secondary kinase domain mutations conferring resistance towards other TKI. Interestingly, crenolanib does not target the wildtype KIT isoform, which is believed to reduce clinical side effects such as prolonged myelosuppression observed with other TKI. Clinical studies are currently enrolling. We now show that gain-of-function mutations of KIT, including codon D816 alterations as the most prevalent mutation in SM and CBFL, sensitize the mutant isoform to crenolanib. Several mast cell and leukemia cell lines harboring autoactivating KIT or FLT3 isoforms were treated with crenolanib in dose dilution series (MOLM14, MV4;11, HMC1.1/1.2, p815). To minimize cell-type specific off-target effects, an isogenic cell model was established. The murine pro B-cell line Ba/F3 was retrovirally transduced with either a FLT3 ITD or a KIT D816 isoform. Apoptosis induction was analyzed by annexin V-based assays. FLT3/KIT tyrosine phosphorylation was assessed by western immunoblots. As previously described, the FLT3 ITD positive cell line MOLM14 revealed high sensitivity towards crenolanib with IC50s in the lowest nanomolar range. We also confirmed high sensitivity towards crenolanib ex vivo in the low nanomolar range in a native sample of a heavily pretreated patient. This patient relapsed with FLT3 ITD positive leukemia harboring a secondary D835H mutation in a subclone. Interestingly, leukemia cells in the relapse situation were much more oncogene-addicted than cells at primary diagnosis, which is in line with previous findings by others. Due to the structural homology of FLT3 D835 and KIT D816 mutations, we extended our studies to mutant-KIT mastocytosis and leukemia cell models and confirm clinically relevant antiproliferative as well as proapoptotic sensitivities towards crenolanib: for HMC mastocytosis cells harboring a KIT V560G and/or a D816V mutation, potent induction of apoptosis was observed with IC50s of 100-250nM. The murine p815 mastocytosis cell line (harboring a D814Y mutation corresponding to D816Y in humans) demonstrated a proapoptotic effect of crenolanib with an IC50 of 60 nM. Treatment of corresponding KIT or FLT3 isoform-transduced Ba/F3 cells confirmed similar IC50s in the leukemia cell lines. Parental Ba/F3cells did not show any sensitivity towards crenolanib up to concentrations of 1000 nM. Additionally, potent dephosphorylation at 100 nM of KIT D816V in Ba/F3 and HMC cells after exposure to crenolanib confirmed mutant-KIT as a target of the drug. Evaluation of a broader range of native mast cell and leukemia patient samples as well as additional leukemia cell lines and isogenic Ba/F3 KIT or FLT3 transfectants is ongoing. First results demonstrate activity of crenolanib in native cells of a subset of patient samples with SM or CBFL treated ex vivo. Even more, combination of crenolanib with anthracyclines revealed additive to superadditive proapoptotic effects. Moreover, combination of crenolanib with cladribine, a hallmark agent in the treatment of systemic mastocytosis, resulted in potent induction of apoptosis already at doses that did not display any proapoptotic effects when administered as single agents, thereby providing a rationale for combinatorial therapeutic approaches. In summary, crenolanib is effective against the KIT D816V isoform associated with several hematologic malignancies. Notably, while not as effective towards mutant-KIT compared to the FLT3 ITD isoform, the observed estimated IC50 of crenolanib is well in the range of achievable plasma concentrations and in the range of the potent KIT inhibitor dasatinib, which is successfully under clinical investigation in CBFL. Our data provide a rationale to test crenolanib as a potent inhibitor of mutant-KIT isoforms in KIT-associated neoplasms. Disclosures Schuster: AROG Pharmaceuticals: Employment. Ramachandran:AROG: Employment.

Introduction Constitutive phosphorylation of AKT is frequently found in acute leukemia. In a proportion of patients, activation of the PI3K/AKT pathway can be linked to gain-of-function tyrosine kinase mutations. We have previously shown that TKI only incompletely inactivate AKT signaling. PI3K/AKT pathway inhibitors are attractive for targeting this pathway. However, most inhibitors lead to G1/G0 arrest. Likely for this reason, response rates to PI3K inhibitor treatment were moderate in various neoplasms. We here demonstrate that the dual PI3K-MTORC1/2 inhibitor NVP-BEZ235, which is currently under clinical investigation in relapsed acute leukemia, mediates a profound G1/G0 arrest in cells harboring leukemia-driving FLT3 ITD or BCR-ABL1 mutations, impairing induction of apoptosis. Furthermore, combination with TKI or chemotherapy overcomes cellular resistance. Methods Proliferation and apoptosis assays were performed in leukemia cell lines, an isogenic Ba/F3 cell model harboring mutant-KIT, FLT3 or ABL1 isoforms and primary leukemic cells as well as healthy donor cells. Immunoblots were performed to study effects on AKT signaling pathways including downstream effectors of autophagy (p-ULK1), proliferation, cell cycle (p70S6Kinases, cyclinD, pRB) and apoptosis (caspases). Isobolograms were created to compute combination indices. Results NVP-BEZ235 profoundly inhibited phosphorylation of AKT and p70S6K, leading to a potent antiproliferative effect in the low nanomolar range in most cell lines as well as patient blasts. However, no meaningful induction of apoptosis was observed. Instead, a sustained G1/G0 cell cycle arrest with dephosphorylation of RB and upregulation of cyclinD, induction of protective autophagy pathways (via ULK1) and absence of cleaved caspase 3 was detected. An isogenic Ba/F3 model confirmed the low proapoptotic efficacy especially for FLT3 ITD and BCR-ABL1. In an attempt to overcome NVP-BEZ235 induced cell cycle arrest, we co-treated leukemic cells with specific TK inhibitors or chemotherapeutics such as daunorubicin in a fixed dose-dilution ratio. Combination indices revealed additive to superadditive proapoptotic effects for all tested combinations. Conclusion We provide evidence that the antileukemic activity of NVP-BEZ235 in acute leukemia with mutations in tyrosine kinases is abrogated due to induction of profound G1/G0 cell cycle arrest which can be overcome by combination with TKI or chemotherapy. Disclosures: No relevant conflicts of interest to declare.

Introduction Activating mutations of the KIT class III receptor tyrosine kinase (TK) are associated with the pathophysiology of acute leukemia, especially core binding factor leukemia (CBFL), and systemic mastocytosis (SM). Despite considerable antiproliferative and proapoptotic activity of several KIT TK inhibitors in vitro, clinical efficacy in AML and SM is generally moderate. We hypothesized that resistance to therapy is promoted by activation of alternative signaling pathways. Previously we reported that KIT TK inhibition results in significantly increased phosphorylation of heat shock protein (HSP) family members and that KIT is a client protein of phosphorylated (p)HSP90 putatively stabilizing KIT protein function in the presence of KIT TK-inhibitors (Kampa-Schittenhelm et al., ASH annual meeting 2010). This prompted us to further test the HSP inhibitor IPI-504 in KIT dependent CBFL, including the leukemic stem cell fraction. Methods Protein expression levels of (p)HSPs in leukemic blasts of high-risk and CBFL patients were studied by flow cytometry focusing on the CD34+/CD38- leukemia stem/progenitor cell fraction. Cellular proliferation and induction of apoptosis in leukemia cells treated with the HSP90 inhibitor IPI-504 was determined by XTT- and annexin V-based assays. Results (p)HSP90/60 levels were preferentially upregulated in CBFL associating with KIT dysregulation. Consequently, HSP90 inhibition with IPI-504 potently degraded KIT expression causing a direct antiproliferative and antiapoptotic effect in CBFL in in vitro and ex vivo models. Efficacy of IPI-504 was potentiated when combined with TK inhibitors. Importantly, high expression of (p)HSP90 and HSP60 was particularly observed in the CD34+/CD38- putative leukemia stem cell fraction arguing for a function as protection mechanism to conserve (leukemic) progenitor cell function upon cell stress such as antileukemic treatment. Conclusion HSPs are upregulated in CBFL, and IPI-504 induces antiproliferative and proapoptotic effects in primary leukemia samples. Importantly, in particular the putative malignant progenitor cell pool in KIT-associated acute leukemia expresses high levels of (p)HSP90, identifying HSP90 inhibition as an attractive novel strategy to overcome the therapy-refractory behavior of malignant stem/progenitor cells. Our results provide a rationale for the evaluation of HSP90 inhibitors such as IPI-504 in CBFL. Disclosures: No relevant conflicts of interest to declare.