Alternative splicing of Apoptosis Stimulating Protein of TP53-2 (ASPP2) results in an oncogenic isoform promoting migration and therapy resistance in soft tissue sarcoma (STS).

BACKGROUND: Metastatic soft tissue sarcoma (STS) are a heterogeneous group of malignancies which are not curable with chemotherapy alone. Therefore, understanding the molecular mechanisms of sarcomagenesis and therapy resistance remains a critical clinical need. ASPP2 is a tumor suppressor, that functions through both p53-dependent and p53-independent mechanisms. We recently described a dominant-negative ASPP2 isoform (ASPP2κ), that is overexpressed in human leukemias to promote therapy resistance. However, ASPP2κ  has never been studied in STS.  MATERIALS AND METHODS: Expression of ASPP2κ was quantified in human rhabdomyosarcoma tumors using immunohistochemistry and qRT-PCR from formalin-fixed paraffin-embedded (FFPE) and snap-frozen tissue. To study the functional role of ASPP2κ in rhabdomyosarcoma, isogenic cell lines were generated by lentiviral transduction with short RNA hairpins to silence ASPP2κ expression. These engineered cell lines were used to assess the consequences of ASPP2κ silencing on cellular proliferation, migration and sensitivity to damage-induced apoptosis. Statistical analyses were performed using Student's t-test and 2-way ANOVA. RESULTS: We found elevated ASPP2κ mRNA in different soft tissue sarcoma cell lines, representing five different sarcoma sub-entities. We found that ASSP2κ mRNA expression levels were induced in these cell lines by cell-stress. Importantly, we found that the median ASPP2κ expression level was higher in human rhabdomyosarcoma in comparison to a pool of tumor-free tissue. Moreover, ASPP2κ levels were elevated in patient tumor samples versus adjacent tumor-free tissue within individual patients. Using isogenic cell line models with silenced ASPP2κ expression, we found that suppression of ASPP2κ enhanced chemotherapy-induced apoptosis and attenuated cellular proliferation. CONCLUSION: Detection of oncogenic ASPP2κ in human sarcoma provides new insights into sarcoma tumor biology. Our data supports the notion that ASPP2κ promotes sarcomagenesis and resistance to therapy. These observations provide the rationale for further evaluation of ASPP2κ as an oncogenic driver as well as a prognostic tool and potential therapeutic target in STS.

Identification of driver and passenger mutations of FLT3 by high-throughput DNA sequence analysis and functional assessment of candidate alleles.

Mutations in the juxtamembrane and kinase domains of FLT3 are common in AML, but it is not known whether alterations outside these regions contribute to leukemogenesis. We used a high-throughput platform to interrogate the entire FLT3 coding sequence in AML patients without known FLT3 mutations and experimentally tested the consequences of each candidate leukemogenic allele. This approach identified gain-of-function mutations that activated downstream signaling and conferred sensitivity to FLT3 inhibition and alleles that were not associated with kinase activation, including mutations in the catalytic domain. These findings support the concept that acquired mutations in cancer may not contribute to malignant transformation and underscore the importance of functional studies to distinguish "driver" mutations underlying tumorigenesis from biologically neutral "passenger" alterations.

Evaluation of apoptosis stimulating protein of TP53-1 (ASPP1/PPP1R13B) to predict therapy resistance and overall survival in acute myeloid leukemia (AML).

ASPP1 (PPP1R13B) belongs to a family of p53-binding proteins and enhances apoptosis by stimulation of p53-transactivation of selected proapoptotic target genes. It is preferentially expressed in hematopoietic stem cells (HSC) and together with p53 preserves the genomic integrity of the HSC pool. Consequently, dysfunction of ASPP1 has been associated with malignant transformation and development of acute lymphoblastic leukemias and lymphomas - whereas methylation of the promoter region is linked to reduced transcription and ultimately attenuated expression of ASPP1. The role of ASPP1 in AML is not known. We now show that impaired regulation of PPP1R13B contributes to the biology of leukemogenesis and primary therapy resistance in AML. PPP1R13B mRNA expression patterns thereby define a distinct prognostic profile - which is not reflected by the European leukemia net (ELN) risk score. These findings have direct therapeutic implications and we provide a strategy to restore ASPP1 protein levels using hypomethylating agents to sensitize cells towards proapoptotic drugs. Prospective clinical trials are warranted to investigate the role of ASPP1 (PPP1R13B) as a biomarker for risk stratification and as a potential therapeutic target to restore susceptibility to chemotherapy.

ASPP2κ Is Expressed In Human Colorectal Carcinoma And Promotes Chemotherapy Resistance And Tumorigenesis.

Alternative splicing is a common physiologic mechanism to generate numerous distinct gene products from one gene locus, which can result in unique gene products with differing important functional outcomes depending on cell context. Aberrant alternative splicing is a hallmark of cancer that can contribute to oncogenesis and aggressiveness of the disease as well as resistance to therapy. However, aberrant splicing might also result in novel targets for cancer therapy. ASPP2 is a haplo-insufficient tumor suppressor, that functions through both p53-dependent as well as p53-independent mechanisms to enhance cell death after stress. Interestingly, the common human tumor TP53 mutations result in a loss of the binding sites to ASPP2, leading to impaired induction of apoptosis. Vice versa, attenuation of ASPP2 has been described to be associated with high-risk disease, therapy failure and poor clinical outcome especially in tumors harboring the TP53 wildtype (WT) isoform. We have recently identified a novel, dominant-negative splicing variant of ASPP2, named ASPP2κ, with oncogenic potential. Exon-skipping results in a reading-frame shift with a premature translation stop, omitting most of the ASPP2 C-terminus - which harbors the p53-binding domain. Consequently, the ASPP2-p53 interaction is abrogated, which in part impacts on oncogenesis, aggressiveness of disease and response to therapy. Since ASPP2κ has been shown in hematologic malignancies to promote tumorigenesis, we further wished to determine if aberrant ASPP2κ expression plays a role in human solid tumors. In this report, we find that ASPP2κ is frequently expressed in human colorectal tumors (CRC). Using ASPP2κ overexpressing and interference CRC models, we demonstrate a functional role of ASPP2κ in contributing to oncogenesis and resistance to therapy in CRC by 1) enhancing proliferation, 2) promoting cell migration and, 3) conferring resistance to chemotherapy induced apoptosis. Our findings have far-reaching consequences for future diagnostic and therapeutic strategies for ASPP2κ expressing colorectal cancer patients and provide proof-of-principle to further explore ASPP2κ as potential predictive marker and target for therapy in clinical trials.

Molecular determinants of response to matuzumab in combination with paclitaxel for patients with advanced non-small cell lung cancer.

Antibodies targeting epidermal growth factor receptor (EGFR) have proven to be effective in patients with non-small cell lung cancer (NSCLC) that express EGFR. We recently published a phase I study of weekly matuzumab plus paclitaxel. This therapy was well tolerated and showed clinical responses in the majority of patients. Although matuzumab displays potent antitumor activity in some patients, not all patients respond well to treatment. Whether dysregulation of EGFR-mediated pathways precludes or sensitizes cells to paclitaxel is unknown. We sought to determine molecular predictive factors for therapy response in a phase I/II study patient cohort treated with matuzumab+/-paclitaxel. Twenty-three cases [including one complete response (CR), three partial responses (PR), 10 stable diseases (SD)] were screened using immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), PCR/sequencing and denaturing wave high performance liquid chromatography (D-HPLC) for expression, amplification, and mutation status of EGFR and downstream signaling pathways. All patients with PR or CR displayed an either high overall or single-cell EGFR expression in the majority of cells. In addition, all of the moderate responders, who achieved SD after at least two cycles of therapy, showed diffuse EGFR expression rates and/or strong single-cell EGFR expression. In contrast, 44% of the nonresponders showed low overall or single-cell EGFR expression levels. No low-expressing EGFR cases were present within the responder group. In addition, among patients with a gain-of-function mutation in KRAS primary therapy failure and/or short responses to therapy were observed. Our data suggest that EGFR expression and KRAS mutation status is predictive for clinical response to matuzumab +/- paclitaxel in patients with advanced NSCLC.

Valproate and Retinoic Acid in Combination With Decitabine in Elderly Nonfit Patients With Acute Myeloid Leukemia: Results of a Multicenter, Randomized, 2 × 2, Phase II Trial.

PURPOSE: DNA-hypomethylating agents are studied in combination with other epigenetic drugs, such as histone deacetylase inhibitors or differentiation inducers (eg, retinoids), in myeloid neoplasias. A randomized, phase II trial with a 2 × 2 factorial design was conducted to investigate the effects of the histone deacetylase inhibitor valproate and all-trans retinoic acid (ATRA) in treatment-naive elderly patients with acute myeloid leukemia (AML). PATIENTS AND METHODS: Two hundred patients (median age, 76 years; range, 61-92 years) ineligible for induction chemotherapy received decitabine (20 mg/m2 intravenously, days 1 to 5) alone (n = 47) or in combination with valproate (n = 57), ATRA (n = 46), or valproate + ATRA (n = 50). The primary endpoint was objective response, defined as complete and partial remission, tested at a one-sided significance level of α = .10. Key secondary endpoints were overall survival, event-free survival, and progression-free survival and safety. RESULTS: The addition of ATRA resulted in a higher remission rate (21.9% with ATRA v 13.5% without ATRA; odds ratio, 1.80; 95% CI, 0.86 to 3.79; one-sided P = .06). For valproate, no effect was observed (17.8% with valproate v 17.2% without valproate; odds ratio, 1.06; 95% CI, 0.51 to 2.21; one-sided P = .44). Median overall survival was 8.2 months with ATRA v 5.1 months without ATRA (hazard ratio, 0.65; 95% CI, 0.48 to 0.89; two-sided P = .006). Improved survival was observed across risk groups, including patients with adverse cytogenetics, and was associated with longer response duration. With valproate, no survival difference was observed. Toxicities were predominantly hematologic, without relevant differences between the 4 arms. CONCLUSION: The addition of ATRA to decitabine resulted in a higher remission rate and a clinically meaningful survival extension in these patients with difficult-to-treat disease, without added toxicity.

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.

Dasatinib overrides the differentiation blockage in a patient with mutant-KIT D816V positive CBFß-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.

Attenuated expression of apoptosis stimulating protein of p53-2 (ASPP2) in human acute leukemia is associated with therapy failure.

Inactivation of the p53 pathway is a universal event in human cancers and promotes tumorigenesis and resistance to chemotherapy. Inactivating p53 mutations are uncommon in non-complex karyotype leukemias, thus the p53-pathway must be inactivated by other mechanisms. The Apoptosis Stimulating Protein of p53-2 (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. Now, we reveal that ASPP2 expression is significantly attenuated in acute myeloid and lymphoid leukemia - especially in patients with an unfavorable prognostic risk profile and patients who fail induction chemotherapy. In line, knock down of ASPP2 in expressing leukemia cell lines and native leukemic blasts attenuates damage-induced apoptosis. Furthermore, cultured blasts derived from high-risk leukemias fail to induce ASPP2 expression upon anthracycline treatment. The mechanisms of ASPP2 dysregulation are unknown. We provide evidence that attenuation of ASPP2 is caused by hypermethylation of the promoter and 5'UTR regions in native leukemia blasts. Together, our results suggest that ASPP2 contributes to the biology of leukemia and expression should be further explored as a potential prognostic and/or predictive biomarker to monitor therapy responses in acute leukemia.

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--resulting in a potential decrease of 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.