The FLT3 inhibitor PKC412 in combination with cytostatic drugs in vitro in acute myeloid leukemia

An internal tandem duplication of FLT3 (FLT3/ITD) occurs in approximately 25% of newly diagnosed AML. PKC412 inhibits the growth of leukemic cell lines with FLT3 mutations such as the MV4-11. This study evaluated the in vitro effects of the combination of PKC412 and ara-C or daunorubicin, studying the effect of co-incubation, pre-incubation and sequential incubation of the drugs in patient samples and cell lines. Thirty-three patients with AML were included. Two cell lines were studied; MV4-11 that expresses the FLT3/ITD and HL-60 that does not. In the patient cells PKC412 exerted its effect at concentrations between 0.1 and 2.0 microM. For MV4-11 cells concentrations down to 1 nM were effective. In patient samples, the results of co-incubation of PKC412 with ara-C were synergistic in 5%, additive in 67%, sub additive in 17% and antagonistic in 11% of the cases. In patient cells, incubations with ara-C and PKC412 resulted in synergistic effects in 17% of the FLT3/ITD positive samples compared to 0% synergistic in the FLT3/ITD negative samples (p < 0.01). Antagonistic effects were more common in the FLT3/ITD negative samples. The timing of the drugs had little impact on the effect. In cell lines, antagonistic effects were seen frequently in HL-60 (90%) and less so in MV4-11 (60%) regardless of sequence or timing of the drugs. The combination of daunorubicin and PKC412 resulted in more synergistic and less antagonistic effects compared to combinations with ara-C, in both patient material and cell lines. The combination of Lonafarnib, a farnesyl-transferase inhibitor (FTI) and PKC412 had additive and synergistic effects in both FLT3/ITD positive and negative cell lines. In conclusion, the combination of PKC412 together with chemotherapeutic drugs is more effective in FLT3/ITD positive AML cells. Antagonistic effects can be seen, especially in patient samples without FLT3/ITD. Also, the combination of PKC412 and the farnesylinhibitor lonafarnib should be further explored.     

FLT3 inhibitor KRN383 on xenografted human leukemic cells harboring FLT3-activating mutations FLT3 in AML: much more to learn about biology and optimal targeting

Aberrant FLT3 function in leukemia blasts is associated with a poor prognosis. A number of FLT3 modulators are in development. FLT3 mutations may synergistize with other molecular abnormalities in myeloid transformation. Further insights into FLT3 biology are needed to optimally study the therapeutic role of FLT3 inhibitors.     

胰腺癌分子靶向治疗进展

胰腺癌在胃肠道肿瘤中预后差,5年生存率不到5％,发病率呈逐渐上升趋势。治疗主要是外科手术、化疗和放疗相结合的综合治疗,但目前常规治疗效果有限,因此针对胰腺癌生物学特性进行治疗是改善预后的关键。随着靶向治疗的进展,VEGF单克隆抗体和EGFR抑制剂在临床试验中都表现出较好的前景。其他靶向药物,如沙利度胺、基质金属蛋白酶抑制剂、COX-2抑制剂和法尼基转移酶抑制剂还在研究之中。分子靶向治疗将为胰腺癌的治疗提供新的机会。     

Systemic therapy for advanced pancreatic cancer

Death from pancreatic cancer remains high with few long-term survivors. Systemic chemotherapy with 5-fluorouracil-based combinations had minimal impact on natural history of this disease. Several new agents with activity against pancreatic cancer have been identified over the past decade. Gemcitabine has modest activity in this disease. Combination chemotherapy trials incorporating gemcitabine, cisplatin, 5-fluorouracil, oxaliplatin, docetaxel or irinotecan show improved outcomes in objective response rates and survival that need to be confirmed in prospectively randomized studies. Advancement in the understanding of the biology of pancreatic cancer has helped identify several molecular targets for the development of novel therapies. Ongoing and future treatment regimens for pancreatic cancer will incorporate traditional cytotoxic drugs and novel targeted therapies.     

Systemic therapy for pancreatic cancer

Pancreatic adenocarcinoma is typically diagnosed at an advanced stage, at which point systemic therapy becomes the primary modality of treatment. Although gemcitabine monotherapy has remained the standard of care for the past decade, a number of large studies conducted over the past several years have suggested that some advantages may be derived from the use of combination therapy, such as the addition of a platinum agent to gemcitabine. The application of pharmacokinetic principles to drug delivery may confer additional benefit as well. Furthermore, with an increasing understanding of the cellular and stromal events that govern pancreatic tumor maintenance, molecularly targeted agents are under active investigation and may prove to serve a critical role in our therapeutic armamentarium. The combination of standard cytotoxic agents and these newer targeted compounds offers promise that we will be able to make significant inroads in improving clinical outcomes for patients with advanced pancreatic cancer.     

Gemcitabine triggers angiogenesis-promoting molecular signals in pancreatic cancer cells: Therapeutic implications

Pancreatic tumor microenvironment (TME) is characterized by poor tumor-vasculature and extensive desmoplasia that together contribute to poor response to chemotherapy. It was recently shown that targeting of TME to inhibit desmoplasiatic reaction in a preclinical model resulted in increased microvessel-density and intratumoral drug concentration, leading to improved therapeutic response. This approach; however, failed to generate a favorable response in clinical trial. In that regard, we have previously demonstrated a role of gemcitabine-induced CXCR4 signaling as a counter-defense mechanism, which also promoted invasiveness of pancreatic cancer (PC) cells. Here, we investigated the effect of gemcitabine on endothelial cell phenotype. Gemcitabine-treatment of human-umbilical-vein-endothelial-cells (HUVECs) did not promote the growth of HUVECs; however, it was induced when treated with conditioned media from gemcitabine-treated (Gem-CM) PC cells due to increased cell-cycle progression and apoptotic-resistance. Moreover, treatment of HUVECs with Gem-CM resulted in capillary-like structure (CLS) formation and promoted their ability to migrate and invade through extracellular-matrix. Gemcitabine-treatment of PC cells induced expression of various growth factors/cytokines, including IL-8, which exhibited greatest upregulation. Further, IL-8 depletion in Gem-CM diminished its potency to promote angiogenic phenotypes. Together, these findings suggest an indirect effect of gemcitabine on angiogenesis, which, in light of our previous observations, may hold important clinical significance.     

PHD3 regulates differentiation, tumour growth and angiogenesis in pancreatic cancer

Purpose:

Tumour hypoxia activates hypoxia-inducible factor-1 (HIF-1) and indluences angiogenesis, cell survival and invasion. Prolyl hydroxylase-3 (PHD3) regulates degradation of HIF-1α. The effects of PHD3 in tumour growth are largely unknown.

Experimental design:

PHD3 expression was analysed in human pancreatic cancer tissues and cancer cell lines by real-time quantitative PCR and immunohistochemistry. PHD3 overexpression was established by stable transfection and downregulation by short interfering RNA technology. VEGF was quantified by enzyme-linked immunosorbent assay. Matrigel invasion assays were performed to examine tumour cell invasion. Apoptosis was measured by annexin-V staining and caspase-3 assays. The effect of PHD3 on tumour growth in vivo was evaluated in an established orthotopic murine model.

Results:

PHD3 was upregulated in well-differentiated human tumours and cell lines, and regulated hypoxic VEGF secretion. PHD3 overexpression mediated tumour cell growth and invasion by induction of apoptosis in a nerve growth factor-dependent manner by the activation of caspase-3 and phosphorylation of focal adhesion kinase HIF-1 independently. In vivo, PHD3 inhibited tumour growth by abrogation of tumour angiogenesis.

Conclusion:

Our results indicate essential functions of PHD3 in tumour growth, apoptosis and angiogenesis and through HIF-1-dependent and HIF-1-independent pathways.     

晚期胰腺癌二线治疗的研究进展

一线使用吉西他滨已成为晚期胰腺癌治疗的标准方案,但是目前尚无标准的二线化疗方案。支持二线治疗优于最佳支持治疗的数据少之又少。我们总结已公布的二线化疗方案,大部分为Ⅱ期临床试验,以摘要的形式发表居多,没有单药的广泛研究。以药物的副作用作为代价,二线化疗的益处是非常有限的,但Ⅲ期随机试验还需要继续进行。未来的二线化疗将不仅以有效率或生存期作为评价,还将把临床受益率作为治疗目标。     

Inhibition of peritoneal dissemination of ovarian cancer by tyrosine kinase receptor inhibitor SU6668 (TSU-68)

SU6668 (TSU-68) is a small-molecule synthetic inhibitor of the angiogenic related receptor tyrosine kinases Flk-1/KDR, PDGFRbeta, and FGFR1. Using a mouse model of peritoneally disseminated ovarian cancer, we investigated whether SU6668 inhibits peritoneal dissemination and prolongs survival time. BALB/c nude mice were intraperitoneally (i.p.) inoculated with SHIN-3 (VEGF-hypersecretory) or KOC-2S (PDGF-hypersecretory) ovarian serous adenocarcinoma cells with marked peritoneal dissemination ability. From the day after i.p. inoculation of tumor cells, SU6668 was orally administered 6 times weekly at a daily dose of 100 mg/kg or 400 mg/kg. The SU6668-administered group and the vehicle-administered control group were compared for the number of tumor vascular endothelial cells, weight of peritoneally disseminated tumors, amount of ascitic fluid and survival time. As a result, these 3 parameters were significantly smaller in the SHIN-3-inoculated, SU6668-administered mice than in the control group (p = 0.03, p = 0.002, and p = 0.02, respectively). The mean survival time was significantly longer, at 58.1 +/- 11.2 days, in the SU6668-administered mice than that (34.5 +/- 8.8 days) in the control group (p = 0.002). Similarly, in the KOC-2S-inoculated mice, the oral administration of SU6668 significantly reduced these 3 parameters (p = 0.04, p = 0.04, and p = 0.03, respectively), and significantly prolonged survival (16.6 +/- 1.7 days vs. 11.0 +/- 0.7 days, p = 0.008). Thus, the oral administration of SU6668 inhibited angiogenesis and peritoneal dissemination and prolonged survival in mice with peritoneally disseminated ovarian cancer. These effects were observed with both the VEGF- and PDGF-hypersecretory cell lines. Our results suggest that molecular targeting with oral SU6668 will become a new therapeutic strategy targeting peritoneally disseminated ovarian cancer.     

Benefit of postoperative adjuvant therapy for pancreatic cancer

BACKGROUND:

Despite the recent completion of several trials of adjuvant therapy after resection for pancreatic adenocarcinoma, the absolute impact on survival and the identification of appropriate patients for treatment has remained controversial. In the current study, the authors sought to identify the impact of adjuvant therapy and factors associated with any improvement in survival after resection of pancreatic cancer.

METHODS:

Through the California Cancer Registry, all California residents diagnosed with pancreatic cancer between 1994 and 2002 were identified. Factors potentially impacting survival were analyzed, including patient demographics, tumor characteristics, and treatment provided. Univariate and multivariate survival analyses were performed by Kaplan‐Meier and Cox regression methods.

RESULTS:

A total of 26,518 patients were identified; 3196 (12.1%) underwent resection as their primary treatment. The median overall survival was 16 months for patients who underwent resection. Prognostic factors associated with better survival included negative lymph node status, well‐differentiated tumors, younger age, female sex, and the receipt of any adjuvant therapy. On multivariate analysis, adjuvant therapy demonstrated a statistically significant, although modest, impact on survival, with a hazards ratio of 0.79 (95% confidence interval, 0.72‐0.87; P < .001). The benefit of adjuvant therapy was only apparent in those patients with lymph node–positive or poorly differentiated tumors.

CONCLUSIONS:

Adjuvant therapy provided for a modest improvement in overall survival after surgical resection of pancreatic cancer. The absolute effect was most pronounced in those patients with poor prognostic indicators. To identify effective systemic therapy for this deadly cancer, future clinical trials of adjuvant therapy should focus on these groups of patients. Cancer 2009. © 2009 American Cancer Society.     

The catalytically defective receptor protein tyrosine kinase EphA10 promotes tumorigenesis in pancreatic cancer cells

EphA10 (erythropoietin‐producing hepatocellular carcinoma receptor A10) is a catalytically defective receptor protein tyrosine kinase in the ephrin receptor family. Although EphA10 is involved in the malignancy of some types of cancer, its role as an oncogene has not been extensively studied. Here, we investigated the influence of EphA10 on the tumorigenic potential of pancreatic cancer cells. Analysis of expression profiles from The Cancer Genome Atlas confirmed that EphA10 was elevated and higher in tumor tissues than in normal tissues in some cancer types, including pancreatic cancer. EphA10 silencing reduced the proliferation, migration, and adhesion of MIA PaCa‐2 and AsPC‐1 pancreatic cancer cells. These effects were reversed by overexpression of EphA10 in MIA PaCa‐2 cells. Importantly, overexpression and silencing of EphA10 respectively increased and decreased the weight, volume, and number of Ki‐67‐positive proliferating cells in MIA PaCa‐2 xenograft tumors. Further, EphA10 expression was positively correlated with invasion and gelatin degradation in MIA PaCa‐2 cells. Moreover, overexpression of EphA10 enhanced the expression and secretion of MMP‐9 in MIA PaCa‐2 cells and increased the expression of MMP‐9 and the vascular density in xenograft tumors. Finally, expression of EphA10 increased the phosphorylation of ERK, JNK, AKT, FAK, and NF‐κB, which are important for cell proliferation, survival, adhesion, migration, and invasion. Therefore, we suggest that EphA10 plays a pivotal role in the tumorigenesis of pancreatic epithelial cells and is a novel therapeutic target for pancreatic cancer.     

Effects of dasatinib on EphA2 receptor tyrosine kinase activity and downstream signalling in pancreatic cancer

Eph receptors constitute the largest family of receptor tyrosine kinases in the human genome. EphA2 is one prominent member that is overexpressed and functionally altered in many invasive cancers, including pancreatic cancer. Dasatinib, which is a multi-targeted kinase inhibitor mainly developed for Bcr-Abl and Src family kinases, has recently been shown to have significant activity against EphA2. As selective small molecule EphA2 inhibitors are not currently available, we investigated the therapeutic potential to target EphA2 by dasatinib in pancreatic cancer cell lines. Using in vitro kinase assays, we found that EphA2 receptor tyrosine kinase was inhibited directly by dasatinib in a dose-dependent manner. Stimulation with ephrinA1 produced rapid increases of EphA2 phosphorylation that were inhibited by dasatinib, although the effects on activation of downstream signalling differed among the pancreatic cancer cell lines. Dasatinib also inhibited ligand-induced binding of EphA2 to the ubiquitin ligase Cbl, and the internalisation and degradation of EphA2, suggesting that these processes are dependent on kinase activity. Treatment with dasatinib decreased EphA2 phosphorylation in BxPC-3 xenografts, suggesting that dasatinib might have activity in pancreatic cancer due to EphA2 inhibition, besides its effects on Src.     

Aurora B kinase inhibitor AZD1152: determinants of action and ability to enhance chemotherapeutics effectiveness in pancreatic and colon cancer

BACKGROUND: AZD1152, the prodrug for AZD1152-hydroxyquinazoline pyrazol anilide (HQPA), is a selective inhibitor of Aurora B kinase activity. Preclinical evaluation of AZD1152 has been reported in several human cancer models. The potentiality of this compound in combination therapy warrants further investigation in solid tumours. EXPERIMENTAL DESIGN: This study explored the effects of AZD1152-HQPA in colon and pancreatic tumour cells. The antitumour properties of AZD1152, either as single agent or in combination with chemotherapeutics, were evaluated in each study model. The efficacy and the toxicity of AZD1152 alone and in combination with gemcitabine were validated in pancreatic tumour xenograft model.Results:AZD1152-HQPA treatment resulted in a dramatic increase of chromosome number, modification of cell cycle and induction of apoptosis. The most effective combination was that with chemotherapeutics given soon after AZD1152 in both tumour cell types. The effectiveness of the sequential schedule of AZD1152 with gemcitabine was confirmed in nude mice bearing MiaPaCa-2 tumours, showing inhibition of tumour volumes and delaying of tumour growth after the interruption of the treatments. Here we show that AZD1152-HQPA enhances oxaliplatin and gemcitabine effectiveness in colon and pancreatic cancer, respectively. First, we provide advances into administration schedules and dosing regimens for the combination treatment in in vivo pancreatic tumour.     

Paclitaxel and CYC3, an aurora kinase A inhibitor, synergise in pancreatic cancer cells but not bone marrow precursor cells

Background:

Amplification of aurora kinase A (AK-A) overrides the mitotic spindle assembly checkpoint, inducing resistance to taxanes. RNA interference targeting AK-A in human pancreatic cancer cell lines enhanced taxane chemosensitivity. In this study, a novel AK-A inhibitor, CYC3, was investigated in pancreatic cancer cell lines, in combination with paclitaxel.

Methods:

Western blot, flow cytometry and immunostaining were used to investigate the specificity of CYC3. Sulforhodamine B staining, time-lapse microscopy and colony-formation assays were employed to evaluate the cytotoxic effect of CYC3 and paclitaxel. Human colony-forming unit of granulocyte and macrophage (CFU-GM) cells were used to compare the effect in tumour and normal tissue.

Results:

CYC3 was shown to be a specific AK-A inhibitor. Three nanomolar paclitaxel (growth inhibition 50% (GI₅₀) 3 nℳ in PANC-1, 5.1 nℳ in MIA PaCa-2) in combination with 1 μℳ CYC3 (GI₅₀ 1.1 μℳ in MIA PaCa2 and 2 μℳ in PANC-1) was synergistic in inhibiting pancreatic cell growth and causing mitotic arrest, achieving similar effects to 10-fold higher concentrations of paclitaxel (30 nℳ). In CFU-GM cells, the effect of the combination was simply additive, displaying significantly less myelotoxicity compared with high concentrations of paclitaxel (30 nℳ; 60–70% vs 100% inhibition).

Conclusion:

The combination of lower doses of paclitaxel and CYC3 merits further investigation with the potential for an improved therapeutic index in vivo.     

Fms样酪氨酸激酶3配体联合CpG和肿瘤抗原治疗肿瘤的研究

背景与目的:Fms样酪氨酸激酶3配体(fms-like tyrosine kinase 3 ligand,FLT3-L)、CpG虽可作为免疫调节剂发挥抗肿瘤作用,但单独用于治疗肿瘤效果不佳.本研究旨在探讨FLT3-L+肿瘤抗原+CpG联合应用对肝癌小鼠的治疗效果及机制,并探讨FLT3-L与GM-CSF在肿瘤治疗效果方面的差异.方法:选择小鼠H22肝癌模型为研究对象,分为FLT3-L+CpG组、GM-CSF+CpG组和PBS组.皮下注射给药后,用流式细胞术检测各组小鼠体内免疫细胞激活情况,免疫组化法检测肿瘤组织及局部细胞浸润情况,ELISA法检测小鼠体液免疫激活情况,同时观察各组小鼠生存期的差异,评价各组药物的抗肿瘤效果及可能的作用机制.结果:(1)FLT3-L+CpG组小鼠肿瘤生长明显被抑制,小鼠生存期显著延长,同GM-CSF+CpG组和PBS组相比差异具有统计学意义(P＜0.05).(2)脾脏淋巴细胞亚群分析显示,FLT3-L+CpG组CD3+T细胞比例显著上升,CD4+、CD8+亚群比例较PBS组与GM-CSF+CpG组均明显上升,CD4+CD25+Treg细胞比例明显下降,差异有统计学意义(P＜0.05).(3)肿瘤组织形态学分析显示,FLT3-L+CpG组可见大片的组织坏死和显著的淋巴细胞浸润.(4)肿瘤局部浸润淋巴细胞表型分析显示,FLT3-L+CpG组肿瘤局部NK细胞、DC细胞、CD8+T细胞明显增加,较各治疗组均有明显变化,与PBS相比差异有统计学意义(P＜0.05).(5)FLT3-L+CpG组与GM-CSF+CpG组小鼠体内细胞因子IFN-y、TNF-α、IL-2、IL-12均明显升高(P＜0.05).结论:FLT3-L+肿瘤抗原+CpG联合应用可发挥明显的抗肿瘤作用.此方案可能通过打破肿瘤免疫耐受,激活和增加循环中T细胞,并趋化NK细胞、DC细胞、CD8+T细胞到肿瘤局部,发挥杀伤肿瘤细胞的作用;同时可活化免疫因子网络,通过细胞免疫和体液免疫共同发挥抗肿瘤作用.     

胰腺癌综合治疗的研究进展

胰腺癌发病隐匿,进展迅速,预后极差,病死率高。手术是主要治疗手段,但因早期诊断困难,大部分患者确诊时已无手术机会。随着新型化疗药物和方案的不断涌现;放疗技术及设备的明显改进和以基因治疗和免疫治疗为代表的生物疗法的发展,胰腺癌的治疗手段更加多样化和正规化。正确运用综合治疗有望改善胰腺癌的预后。     

Adjuvant therapy of pancreatic cancer

Pancreatic adenocarcinoma is one of the most aggressive tumors, with a high potential for early dissemination and a relatively poor sensitivity to radiation therapy and cytotoxic agents. Complete resection of the tumor is currently the only curative option but only 10–15% of patients present with localized, potentially resectable disease at the time of diagnosis. Median overall survival for all resected patients (R0 and R1) averages between 11 and 23 months, 5-year overall survival ranges from 10 to 25% (R0) and 0 to 5% (R1), leading to a case–fatality index of 95%. Despite the latest trend toward adjuvant chemotherapy with gemcitabine due to the results from the Charité Onkologie-001 trial, there is no broad consensus regarding the adjuvant regimen that should be applied. Early data from the European Study Group for Pancreatic Cancer-3(v2) trial revealed no difference in terms of overall survival between 5-fluorouracil/folinic acid and gemcitabine after resection of pancreatic cancer.