ALBERT

Description: 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.

Description: 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.

Description: Introduction Inactivation of the p53 pathway is a frequent event in human cancers promoting tumorigenesis and resistance to chemotherapy. Inactivating p53 mutations are uncommon in non-complex karyotype leukemias suggesting that the p53-pathway must be inactivated by other mechanisms. ASPP2 is a damage-inducible p53-binding protein that enhances apoptosis at least in part through a p53-mediated pathway. We have previously shown that ASPP2 is an independent haploinsufficient tumor suppressor in vivo and that ASPP2 expression is attenuated in a proportion of acute leukemia patients. We now provide evidence that attenuation of ASPP2 is exclusively linked to patients failing to obtain complete remission after induction chemotherapy. Furthermore, we link attenuated ASPP2 expression to hypermethylation of the promoter and 5’UTR regions of ASPP2. Methods ASPP2 levels were determined using standard qRT-PCR assays as well as FACS and immunoblot analyses. Expression levels in bone marrow samples of 51 leukemia patients were normalized to a control cohort comprising bone marrow samples of 17 different healthy donors. Statistical analyses were performed using the non-parametric Wilcoxon rank-sum test and Kruskal-Wallis test. To silence ASPP2 expression, siRNA experiments were performed using leukemia cell lines and primary leukemic patient blasts. gDNA methylation arrays were performed using patient derived leukemia cells. Results ASPP2 mRNA expression was found to be significantly lower (p=0.02) in acute myeloid (AML) and lymphoid leukemia (ALL) samples (n=51) compared to mononuclear cells from healthy bone marrow donors (n=17). Notably, lower ASPP2 mRNA and protein expression levels were statistically significantly (p=0.02) associated with clinical unfavorable disease according to ELN. We further tested whether ASPP2 levels differ between patients responding or not to induction chemotherapy and found significantly lower overall ASPP2 expression levels in the non-responder cohort (p=0.01). Importantly, 27% of the patients in the non-responding cohort displayed ASPP2 levels below 0.8 (on a normalized scale with the levels in healthy donors set to 1). Such low levels were not observed in good risk patients or in higher-risk patients achieving complete remission after the first cycle of induction chemotherapy. Receiver operating characteristic (ROC) analysis determined

Description: 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.

Description: Inactivation of the p53 pathway is an universal event in human cancers. As p53 mutations are rare in de novo acute leukemias, the p53-pathway must be inactivated by other mechanisms instead. The Apoptosis stimulating Protein of p53-2 (ASPP2) is a haploinsufficient tumor suppressor, which we have shown to be attenuated in acute leukemia. We now provide evidence that ASPP2 regulates pathways involved in early hematopoiesis, and attenuated expression perturbs malignant transformation. Gene-chip microarrays were performed on ASPP2+/+ versus +/- mouse embryonic fibroblasts (MEFs) (-/- knockouts are not viable) and pathway analysis was performed using Affimetrix software. ASPP2 expression was silenced in IL3-dependent Ba/F3 pro-B cells and leukemia cell lines using standard siRNA protocols. Cellular proliferation was assessed using XTT-based assays. To evaluate for malignant transformation, long term cell cultures with stably retrovirally silenced Ba/F3 cells (+/- stress inforcement using gamma irradiation, 4x 5Gy) were employed. IL3 weaning was performed as an indicator of autoactivated cellular proliferation, and analysis of chromosomal defects or gene mutations were performed. Microarray mRNA analysis revealed that attenuated ASPP2 expression resulted in significant alterations of pathways involved in mediating cellular growth, proliferation and tissue differentiation. Subanalysis proofed involvement of genes playing a major role in embryogenesis, the development of the hematopoietic system and leukemogenesis (homeobox (HOX) family members, NOTCH, BCL, IRF7 and EGR1). Population doubling times were significantly increased in ASPP2+/- MEFs compared to the parental cells. Modeling ASPP2 attenuation via specific siRNA knockdown in leukemia cell lines (K562 and Kasumi1) resulted in significantly increased cellular proliferation. Similarly, ASPP2-interference in pro-B Ba/F3 cells lead to perturbed proliferation and an increase of polyploid cells with a dramatic change in morphology with megaloid cells, indicating mitotic failure. This effect was even more pronounced after stress induction with daunorubicin. Lentiviral shRNA-ASPP2 stable knock-out was performed in Ba/F3 cells to follow cells in long term cultures for malignant transformation. Again, we confirmed signs of mitotic failure as observed in the siRNA approach. Even more, gamma irradiated ASPP2-lacking strains were successfully IL3 weaned, which indicates autoactivated cellular proliferation. A screen for acquired oncogenic mutations was performed and analyses are ongoing. In summary, we provide evidence that ASPP2 has a pivotal role in early hematopoiesis, and loss of ASPP2 is a driver mechanism to fuel leukemogenesis. Disclosures No relevant conflicts of interest to declare.

Description: Apoptosis seems to be a major means by which p53 suppresses tumorigenesis in hematopoietic cells. While loss-of-function mutations in p53 are frequently found in most neoplasms, non-complex karyotype acute leukemias are typically p53 wildtype and the p53-pathways are thought to be compromised through other mechanisms. ASPP2 is an independent haploinsufficient tumor suppressor which initiates induction of apoptosis after cellular damage in a p53 dependent manner (Kampa et al., PNAS 2009). We and others have found that attenuation/loss of ASPP2 is a common event in tumors. Here we report the identification of three novel splice variants of ASPP2, which are frequently found in acute leukemias: Tantalizingly, all variants lack the functionally relevant p53 binding sites. The most prevalent isoform lacking exon 17 was termed ASPP2κ and detected in 〉30% of 80 investigated acute leukemia patients. Another variant termed ASPP2μ lacks exon 16/17 – and a third variant, ASPP2λ, lacks exon 17 and most of exon 18. Quantitative PCR on genomic DNA comparing exon 15 and exon 17 expression levels revealed that the variants derived from alternative splicing rather than mutation. All truncated isoforms were found to result in a reading frame-shift with a premature translation stop causing loss of most of the C-terminus harboring the p53-binding sites. Development of a highly sensitive isoform-specific qRT-PCR assay for ASPP2κ revealed low to none mRNA levels in physiologic mononuclear bone marrow cells – In contrast, high ASPP2κ mRNA levels were exclusively found in leukemia blasts. Analysis of the CD34+ fraction confirmed ASPP2κ-expression in leukemia stem/progenitor cells. In addition, analysis of samples derived from patients before and after induction chemotherapy revealed absence of ASPP2κ, resp. ASPP2μ, in patients achieving complete remission after hematopoietic reconstitution, again demonstrating leukemia-specificity of the truncated isoforms. We next generated isoform-specific antibodies for ASPP2κ directed to the hypothetical fusion site and confirmed their sequence-specificity by BlastN search. Subsequent co-immunoprecipitation experiments revealed genuine translation of the splice variants into protein isoforms. Comparative analysis of primary leukemia cells and healthy blood and bone marrow donors demonstrated that ASPP2κ protein is specifically expressed in leukemia. Preliminary functional analysis revealed that ASPP2κ is damage-inducible, suggesting a role of aberrant splicing of ASPP2 in response to cellular stress. Studies of the promoter region suggest alternative/aberrant promoter activation, which was not seen in physiologic cells. Transfection of ASPP2κ in murine pro-B Ba/F3 cells induced a more aggressive phenotype with mitotic failure, perturbed cellular proliferation and attenuated induction of apoptosis upon diverse cellular stressors. Expression of aberrant ASPP2 splice variants might be a underlying initiating events of tumorigenesis. In conclusion, we identified three novel ASPP2 splice variants impairing proapoptotic signaling in native acute leukemia cells. Our data so far suggest aberrant ASPP2 signaling as an initiating event in oncogenesis, giving rise to genomic instability and mutations thereby triggering the actual leukemic clone. Further functional analyses are ongoing. Disclosures: No relevant conflicts of interest to declare.

Description: Background Stargardt disease (SD) is characterized by the accumulation of the age-pigment lipofuscin in the retinal pigment epithelium (RPE) and subsequent neuroretinal degeneration. The disease leads to vision loss early in life. Here, we investigate age-dependent ultrastructural changes in three SD mouse models: albino Abca4-/- and pigmented Abca4-/- and Abca4-/-.Rdh8-/- mice. Since we found indications for oxidative stress primarily in albino SD mice, we tested RPE melanin for its antioxidative capabilities. Methods SD mouse eyes were investigated by light, fluorescence and electron microscopy and were compared to the respective albino and pigmented wild type mice and to a human donor SD eye. To confirm the role of RPE melanin in scavenging oxidative stress, melanin from S. officinalis as a standard and porcine RPE were tested for their capability to quench superoxide anions. Results Histological alterations indicative of oxidative stress and/or lysosomal dysfunction were present in albino Abca4-/- and Abca4-/-.Rdh8-/- mice. Retinal damage, such as inner segment rupture and pyknotic or free photoreceptor nuclei in the subretinal space and RPE vacuolization were exclusively found in albino Abca4-/- mice. Shortened and disorganized photoreceptor outer segments and dead RPE cells were found in albino Abca4-/- and Abca4-/-.Rdh8-/- mice, with earlier onset in albino Abca4-/- mice. Undegraded phagosomes and lipofuscin accumulation were present in the RPE of all three SD strains, but numbers were highest in Abca4-/-.Rdh8-/- mice. Lipofuscin morphology differed between SD strains: (melano-)lipofuscin granules in pigmented Abca4-/- mice had a homogenous electron density and sharp demarcations, while lipofuscin in albino Abca4-/- mice had a flocculent electron density and often lacked a surrounding membrane, indicating loss of lysosomal integrity. Young Abca4-/-.Rdh8-/- mice showed (melano-)lipofuscin granules with homogenous electron density, while in aged animals granules with flocculent electron density predominated. Both strains of pigmented SD mice had melanolipofuscin clusters as found in the human SD eye. Like melanin from S. officinalis, porcine RPE melanin can also quench superoxide anions. Discussion The presented pathologies in albino Abca4-/- and Abca4-/-.Rdh8-/- mice suggest oxidative stress and/or lysosomal dysfunction within the RPE. Since albino Abca4-/- mice have the earliest onset and severest damage and as absence of melanin and also melanin turnover with age are known to diminish RPEs anti-oxidative properties, we assume that RPE melanin plays a role in SD related damages. A lack of pathology in pigmented Abca4-/- mice due to lower stress levels as compared to the Abca4-/-.Rdh8-/- mice underlines this hypothesis. It is also supported by the finding that RPE melanin can quench superoxide anions. We therefore suppose that RPE melanin is important in retinal health and we discuss its role as an oxidative stress scavenger.