Alternative splicing of the tumor suppressor ASPP2 results in a stress-inducible, oncogenic isoform prevalent 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 ASPP2κ 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.

Crenolanib is a type I tyrosine kinase inhibitor that inhibits mutant KIT D816 isoforms prevalent in systemic mastocytosis and core binding factor leukemia.

Activating D816 mutations of the class III receptor tyrosine kinase KIT are associated with the majority of patients with systemic mastocytosis (SM), but also core binding factor (CBF) AML, making KIT mutations attractive therapeutic targets for the treatment of these cancers. Crenolanib is a potent and selective inhibitor of wild-type as well as mutant isoforms of the class III receptor tyrosine kinases FLT3 and PDGFRα/ß. Notably, crenolanib inhibits constitutively active mutant-FLT3 isoforms resulting from amino acid substitutions of aspartic acid at codon 835, which is homologous to codon 816 in the KIT gene - suggesting sensitivity against mutant-KIT D816 isoforms as well. Here we demonstrate that crenolanib targets KIT D816 in SM and CBF AML models: crenolanib inhibits cellular proliferation and initiates apoptosis of mastocytosis cell lines expressing these mutations. Target-specificity was confirmed using an isogenic cell model. In addition, we demonstrate that KIT D816 mutations are targetable with clinically achievable doses of crenolanib. Further, a rationale to combine cladribine (2-CDA), the therapeutic standard in SM, with crenolanib is provided. In conclusion, we demonstrate that crenolanib is an inhibitor of mutant-KIT D816 isoforms at clinically achievable concentrations, and thus may be a potential treatment for SM and CBF AML as a monotherapy or in combination approaches.

Dronabinol has preferential antileukemic activity in acute lymphoblastic and myeloid leukemia with lymphoid differentiation patterns.

BACKGROUND: It 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. METHODS: To 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. RESULTS: Meaningful 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. CONCLUSION: Our 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.

Cell cycle-dependent activity of the novel dual PI3K-MTORC1/2 inhibitor NVP-BGT226 in acute leukemia.

BACKGROUND: Dysregulation 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. METHODS: Native 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. RESULTS: We 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. CONCLUSIONS: Our 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.

Quizartinib (AC220) is a potent second generation class III tyrosine kinase inhibitor that displays a distinct inhibition profile against mutant-FLT3, -PDGFRA and -KIT isoforms.

BACKGROUND: Activating 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. METHODS: Various 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. RESULTS: Kinase 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. CONCLUSION: Quizartinib is a potent second-generation class III receptor TK-inhibitor--but specific, mutation restricted spectrum of activity may require mutation screening prior to therapy.