Marcus M. Schittenhelm

Dasatinib (BMS-354825), a Dual SRC/ABL Kinase Inhibitor, Inhibits the Kinase Activity of Wild-Type, Juxtamembrane, and Activation Loop Mutant KIT Isoforms Associated with Human Malignancies

Activating mutations of the activation loop of KIT are associated with certain human neoplasms, including the majority of patients with systemic mast cell disorders, as well as cases of seminoma, acute myelogenous leukemia (AML), and gastrointestinal stromal tumors (GISTs). The small-molecule tyrosine kinase inhibitor imatinib mesylate is a potent inhibitor of wild-type (WT) KIT and certain mutant KIT isoforms and has become the standard of care for treating patients with metastatic GIST. However, KIT activation loop mutations involving codon D816 that are typically found in AML, systemic mastocytosis, and seminoma are insensitive to imatinib mesylate (IC50 > 5-10 micromol/L), and acquired KIT activation loop mutations can be associated with imatinib mesylate resistance in GIST. Dasatinib (formerly BMS-354825) is a small-molecule, ATP-competitive inhibitor of SRC and ABL tyrosine kinases with potency in the low nanomolar range. Some small-molecule SRC/ABL inhibitors also have potency against WT KIT kinase. Therefore, we hypothesized that dasatinib might inhibit the kinase activity of both WT and mutant KIT isoforms. We report herein that dasatinib potently inhibits WT KIT and juxtamembrane domain mutant KIT autophosphorylation and KIT-dependent activation of downstream pathways important for cell viability and cell survival, such as Ras/mitogen-activated protein kinase, phosphoinositide 3-kinase/Akt, and Janus-activated kinase/signal transducers and activators of transcription. Furthermore, dasatinib is a potent inhibitor of imatinib-resistant KIT activation loop mutants and induces apoptosis in mast cell and leukemic cell lines expressing these mutations (potency against KIT D816Y >> D816F > D816V). Our studies suggest that dasatinib may have clinical efficacy against human neoplasms that are associated with gain-of-function KIT mutations.

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

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.

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

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.

A phase I/II study of sunitinib and intensive chemotherapy in patients over 60 years of age with acute myeloid leukaemia and activating FLT3 mutations.

Acute myeloid leukaemia (AML) with FLT3 mutation has a dismal prognosis in elderly patients. Treatment with a combination of FLT3 inhibitors and standard chemotherapy has not been extensively studied. Therefore, we instigated a phase I/II clinical trial of chemotherapy with cytosine arabinoside (Ara‐C)/daunorubicin induction (7+3) followed by three cycles of intermediate‐dose Ara‐C consolidation in 22 AML patients with activating FLT3 mutations. Sunitinib was added at predefined dose levels and as maintenance therapy for 2 years. At dose level 1, sunitinib 25 mg daily continuously from day 1 onwards resulted in two cases with dose‐limiting toxicity (DLT), prolonged haemotoxicity and hand‐foot syndrome. At dose level −1, sunitinib 25 mg was restricted to days 1–7 of each chemotherapy cycle. One DLT was observed in six evaluable patients. Six additional patients were treated in an extension phase. Thirteen of 22 patients (59%; 8/14 with FLT3–internal tandem duplication and 5/8 with FLT3‐tyrosine kinase domain) achieved a complete remission/complete remission with incomplete blood count recovery. For the 17 patients included at the lower dose level, median overall, relapse‐free and event‐free survival were 1·6, 1·0 and 0·4 years, respectively. Four out of five analysed patients with relapse during maintenance therapy lost their initial FLT3 mutation, suggesting outgrowth of FLT3 wild‐type subclones.

The FLT3 inhibitor tandutinib (formerly MLN518) has sequence-independent synergistic effects with cytarabine and daunorubicin

AML remains a difficult disease to treat. Despite response to induction chemotherapy, most patients ultimately relapse. Further, among elderly patients, aggressive therapy options are often limited due to other medical conditions and decreased tolerance of these patients to conventional chemotherapy. Internal tandem duplications (ITD) of the FLT3 juxtamembrane domain occur in 20-30% of AML patients and predict poor outcome. First clinical data with the FLT3 inhibitor tandutinib demonstrated antileukemic activity in approximately half of the patients - predominantly with FLT3 ITD-positive AML. But the data also show that optimal use of tandutinib will require combination therapy with cytotoxic agents. Notably, single agent tandutinib has not been associated with myelosuppression, mucositis or cardiac toxicity - the dose limiting toxicities of AML chemotherapy. We determined the feasibility of combining tandutinib with the standard “3+7” induction regimen in AML and show that, in contrast to other structurally unrelated FLT3 inhibitors recently evaluated in clinical trials, the use of tandutinib displayed application sequence independent synergistic antileukemic effects in combination with cytarabine and daunorubicin. Strong synergistic antiproliferative and proapoptotic effects were thereby predominantly seen on FLT3 ITD-positive blasts. Further we demonstrate, that addition of tandutinib may allow dose reduction of chemotherapy without loss of overall antileukemic activity – but with a resultant decrease of potential side effects. This approach might be an interesting novel strategy especially in the treatment of elderly/comorbid patients. Our data provide a rationale for combining tandutinib with induction chemotherapy in intensified as well as in dose reduction protocols for FLT3 ITD-positive AML.

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

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.

Dasatinib overrides the differentiation blockage in a patient with mutant- KIT D816V positive CBFβ-MYH11 leukemia

Activating KIT D816V mutations are frequently found in CBF AML, which predicts for an unfavorable outcome. Dasatinib is a potent inhibitor of wildtype and mutant-KIT isoforms – including D816V. We now provide proof of antileukemic efficacy in a patient with relapsing mutant-KIT D816V CBF AML. Importantly, this effect is mediated via overriding the differentiation blockage of the leukemia clone. In addition, we show that dasatinib is capable to induce pulmonary differentiation syndrome – and therefore needs close monitoring of patients under therapy.

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/β. 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.

Abstract LB-49: Identification of C-terminal truncated splice variants of the apoptosis-stimulating protein of p53-2 (ASPP2) in acute leukemia.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC ASPP2 is an independent haploinsufficient tumor suppressor (Kampa et al., PNAS2009) and studies in tumors including breast cancer and aggressive lymphoma have shown that attenuation/loss of ASPP2 is a common occurrence. In acute leukemia, we have evidence that attenuation of ASPP2 significantly associates with higher-risk disease and poor clinical outcome (Schittenhelm et al.,ASH2012). In an attempt to screen for ASPP2 gene/transcript aberrations we identified two novel splicing variants of ASPP2 in acute myeloid and lymphoid leukemia blasts from 80 consented patients: The more prevalent isoform lacking exon 17, named ASPP2κ, was detected in >30% of patients. ASPP2μ, lacking ex16/17, was identified in one patient. A quantitative (q)PCR assay on genomic DNA to compare exon 15 versus exon 17 expression levels did not reveal any significant differences arguing for a splicing event rather than mutation. Isolating CD34(+) leukemia blasts using cell sorting technology demonstrated ASPP2κ-expression in leukemia stem/progenitor cells. Lack of exon 16 (resp. exon 16/17) results in a reading frame-shift with a premature translation stop, omitting most of the C-terminus - which harbors the p53-binding sites. We generated isoform-specific antibodies directed against the hypothetical fusion site. Sequence-specificity was confirmed by BlastN search. Co-immunoprecipitation using antibodies targeting the ASPP2 N-terminal and probing for ASPP2κ-specific antibodies revealed genuine translation into two protein isoforms (ASPP2κ-1/2, corresponding to ASPP2-1/2) with the predicted sizes of 97 and 111 kDa. Western immunoblotting and intracellular semi-quantitative immunophenotyping of freshly isolated native leukemia as well as bone marrow donor samples demonstrated ASPP2κ-1/2 to be specifically expressed in leukemia. Additionally we developed a highly isoform—specific qRT-PCR assay running on a light-cycler platform: A restriction motif-specific ASPP2 exon 17 digest was performed to deplete “wildtype” ASPP2 and ASPP2κ-specific primer were generated. Although ASPP2κ amplicons were detectable in physiologic cells, high expression levels were exclusively found in leukemic blasts. Tantalizingly ASPP2κ as well as ASPP2μ dramatically decreased in patients achieving complete remission (CR) - whereas patients failing remission retained high transcript levels. Preliminary data suggest, that transfection of ASPP2κ in murine pro-B Ba/F3 cells renders cells to more aggressive biology with mitotic failure and perturbed cellular proliferation. Systematic analysis of other tumor entities and functional analysis including other models are ongoing. In summary, we identified two novel ASPP2 splice variants lacking the p53-binding site - which are predominantly detected in acute leukemia and may link to early tumorigenesis. Citation Format: Kerstin M. Kampa-Schittenhelm, Charles D. Lopez, Barbara Illing, Michael Walter, Marcus M. Schittenhelm. Identification of C-terminal truncated splice variants of the apoptosis-stimulating protein of p53-2 (ASPP2) in acute leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-49. doi:10.1158/1538-7445.AM2013-LB-49

Abstract 2238: Role of phosphoinositide-dependent protein kinase-1 (PDK1) in leukemogenesis of acute leukemia

The Phosphoinositide-dependent Protein Kinase-1 (PDK1) is a key regulator of AKT signaling by phosphorylation of a threonine residue at codon 308. PDK1 itself is activated by PI3kinases and plays an important role in growth factor signaling. We previously demonstrated that AKT is globally activated in acute leukemia, e.g. via gain-of function tyrosine kinase mutations. Consecutively we have shown that tyrosine kinase inhibitors suppress codon 473 phosphorylation - but leave Thr308 activated, hypothetically via activation by growth factors. Accordingly, we show that phosphorylation status of AKT in bone marrow leukemia blasts is higher compared to circulating blasts, which favors the hypothesis of a protective bone marrow microenvironment due to the vast occurrence of growth factors and chemokines, which activate PI3K/AKT signaling. We aimed to investigate whether activation of AKT(Thr308) is crucial for the maintenance of leukemia cells and leukemogenesis and silenced PDK1 by retrovirally transducing different short hairpin RNA constructs cloned into a mammalian vector into AML cell lines. Proliferation capacity of the PDK1-silenced cells in comparison to an empty vector control was studied using an XTT-based assay. Cell lines with suppressed transcription of PDK1 revealed a significantly diminished proliferation capacity compared to the empty vector controls. To evaluate potency of tumor engraftment and aggressiveness of disease in vivo, we transplanted stably transfected PDK1-shRNA silenced acute leukemia cells into NOD/SCID mice. Isogenic cells transfected with an empty vector were used as the control group. Mice were regularly monitored for disease symptoms and leukemic blast count. Already on day 8, the peripheral blast count in the control mice revealed a faster increase with higher cell counts (∼ 4 x 10ex3/microL) compared to the PDK1-silenced group (∼ 0,5 x 10ex3/microL). Over time, all mice finally developed leukemia, with the control mice to be the first. In addition, we tested two novel PI3K/mTOR inhibitors, NVP-BEZ235 and NVP-BGT226, with regard to the antiproliferative capacity in ex vivo leukemia blasts. Both agents revealed potent antiproliferation with NVP-BGT226 the more potent agent. NVP-BGT226 induced apoptosis in ex vivo blasts in a subset of patients, with an IC50 in the low nanomolar range. In summary, we here identify the PDK1-AKT signal transduction pathway to be crucial for aggressiveness of disease. Of note, it was not possible to suppress development of leukemia completely, arguing for a multistep mechanism of leukemogenesis. Our data supports descriptive expression analysis of PDK1 or AKT(Thr308) that link high expression with unfavorable clinical outcome. Therefore, targeting the PI3K/AKT axis which activates PDK1 in acute leukemia may be beneficial to improve therapy outcome in a subset of patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2238. doi:1538-7445.AM2012-2238