Activating JAK-mutations confer resistance to FLT3 kinase inhibitors in FLT3-ITD positive AML in vitro and in vivo.

An important limitation of FLT3 tyrosine kinase inhibitors (TKIs) in FLT3-ITD positive AML is the development of resistance. To better understand resistance to FLT3 inhibition, we examined FLT3-ITD positive cell lines which had acquired resistance to midostaurin or sorafenib. In 6 out of 23 TKI resistant cell lines we were able to detect a JAK1 V658F mutation, a mutation that led to reactivation of the CSF2RB-STAT5 pathway. Knockdown of JAK1, or treatment with a JAK inhibitor, resensitized cells to FLT3 inhibition. Out of 136 patients with FLT3-ITD mutated AML and exposed to FLT3 inhibitor, we found seven different JAK family mutations in six of the cases (4.4%), including five bona fide, activating mutations. Except for one patient, the JAK mutations occurred de novo (n = 4) or displayed increasing variant allele frequency after exposure to FLT3 TKI (n = 1). In vitro each of the five activating variants were found to induce resistance to FLT3-ITD inhibition, which was then overcome by dual FLT3/JAK inhibition. In conclusion, our data characterize a novel mechanism of resistance to FLT3-ITD inhibition and may offer a potential therapy, using dual JAK and FLT3 inhibition.

Optimization of the Linker Domain in a Dimeric Compound that Degrades an r(CUG) Repeat Expansion in Cells.

RNA repeat expansions are responsible for more than 30 incurable diseases. Among them is myotonic dystrophy type 1 (DM1), the most common form of adult on-set muscular dystrophy. DM1 is caused by an r(CUG) repeat expansion [r(CUG)exp] located in the 3' untranslated region (UTR) of the dystrophia myotonica protein kinase gene. This repeat expansion is highly structured, forming a periodic array of 5'CUG/3'GUC internal loop motifs. We therefore designed dimeric compounds that simultaneously bind two of these motifs by connecting two RNA-binding modules with peptoid linkers of different geometries and lengths. The optimal linker contains two proline residues and enhances compound affinity. Equipping this molecule with a bleomycin A5 cleaving module converts the simple binding compound into a potent allele-selective cleaver of r(CUG)exp. This study shows that the linker in modularly assembled ligands targeting RNA can be optimized to afford potent biological activity.

Erlotinib is effective against FLT3-ITD mutant AML and helps to overcome intratumoral heterogeneity via targeting FLT3 and Lyn.

Erlotinib has potential therapeutic effect on acute myeloid leukemia (AML) in patients, but the mechanism is not clear. Effective tumor biomarkers for erlotinib in the treatment of AML remain poorly defined. Here, we demonstrate that erlotinib in vitro significantly inhibits the growth of the FLT3-ITD mutant AML cell MV4-11 and Ba/F3-FLT3-ITD cell via targeting FLT3, a certified valid target for the effective treatment of AML. In vivo, oral administration of erlotinib at 100 mg/kg/day induced rapid MV4-11 tumor regression and significantly prolonged the survival time of bone marrow engraftment AML mice via inhibiting the FLT3 signal. Thus, the therapeutic benefits of erlotinib on AML are due to its ability to target FLT3. FLT3-ITD mutation is an effective biomarker for erlotinib during AML treatment. In addition, we also demonstrate that erlotinib inhibits the activity of AML cell KG-1 (no FLT3 expression) by targeting Lyn. Recently, single cell analysis demonstrated that intratumoral heterogeneity are one of the contributors in the relapse and FLT3 inhibitor resistance. Erlotinib could effectively inhibit the MV4-11 cells via targeting FLT3, and inhibit KG-1 cells via targeting Lyn. Therefore, Erlotinib also has the potential to overcome intratumoral heterogeneity via targeting FLT3 and Lyn.

Comparative analysis of epigenetic inhibitors reveals different degrees of interference with transcriptional gene silencing and induction of DNA damage.

Repetitive DNA sequences and some genes are epigenetically repressed by transcriptional gene silencing (TGS). When genetic mutants are not available or problematic to use, TGS can be suppressed by chemical inhibitors. However, informed use of epigenetic inhibitors is partially hampered by the absence of any systematic comparison. In addition, there is emerging evidence that epigenetic inhibitors cause genomic instability, but the nature of this damage and its repair remain unclear. To bridge these gaps, we compared the effects of 5-azacytidine (AC), 2'-deoxy-5-azacytidine (DAC), zebularine and 3-deazaneplanocin A (DZNep) on TGS and DNA damage repair. The most effective inhibitor of TGS was DAC, followed by DZNep, zebularine and AC. We confirmed that all inhibitors induce DNA damage and suggest that this damage is repaired by multiple pathways with a critical role of homologous recombination and of the SMC5/6 complex. A strong positive link between the degree of cytidine analog-induced DNA demethylation and the amount of DNA damage suggests that DNA damage is an integral part of cytidine analog-induced DNA demethylation. This helps us to understand the function of DNA methylation in plants and opens the possibility of using epigenetic inhibitors in biotechnology.

Rationally Designed Anti-CRISPR Nucleic Acid Inhibitors of CRISPR-Cas9.

Clustered regularly interspaced short palindromic repeat (CRISPR) RNAs and their associated effector (Cas) enzymes are being developed into promising therapeutics to treat disease. However, CRISPR-Cas enzymes might produce unwanted gene editing or dangerous side effects. Drug-like molecules that can inactivate CRISPR-Cas enzymes could help facilitate safer therapeutic development. Based on the requirement of guide RNA and target DNA interaction by Cas enzymes, we rationally designed small nucleic acid-based inhibitors (SNuBs) of Streptococcus pyogenes (Sp) Cas9. Inhibitors were initially designed as 2'-O-methyl-modified oligonucleotides that bound the CRISPR RNA guide sequence (anti-guide) or repeat sequence (anti-tracr), or DNA oligonucleotides that bound the protospacer adjacent motif (PAM)-interaction domain (anti-PAM) of SpCas9. Coupling anti-PAM and anti-tracr modules together was synergistic and resulted in high binding affinity and efficient inhibition of Cas9 DNA cleavage activity. Incorporating 2'F-RNA and locked nucleic acid nucleotides into the anti-tracr module resulted in greater inhibition as well as dose-dependent suppression of gene editing in human cells. CRISPR SNuBs provide a platform for rational design of CRISPR-Cas enzyme inhibitors that should translate to other CRISPR effector enzymes and enable better control over CRISPR-based applications.

Synthesis and characterization of amino acid substituted sunitinib analogues for the treatment of AML.

Acute myeloid leukemia (AML) is the most common type of leukemia in adults. Sunitinib, a multikinase inhibitor, was the first Fms-like tyrosine kinase 3 (FLT3) inhibitor clinically used against AML. Off-target effects are a major concern for multikinase inhibitors. As targeted delivery may reduce such undesired side effects, our goal was to develop novel amino acid substituted derivatives of sunitinib which are potent candidates to be used conjugated with antibodies and peptides. In the current paper we present the synthesis, physicochemical and in vitro characterization of sixty two Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutant kinase inhibitors, bearing amino acid moieties, fit to be conjugated with peptide-based delivery systems via their carboxyl group. We determined the solubility, pKa, CHI and LogP values of the compounds along with their inhibition potential against FLT3-ITD mutant kinase and on MV4-11 cell line. The ester derivatives of the compounds inhibit the growth of the MV4-11 leukemia cell line at submicromolar concentration.

Tyrosine kinase inhibition increases the cell surface localization of FLT3-ITD and enhances FLT3-directed immunotherapy of acute myeloid leukemia.

The fms-related tyrosine kinase 3 (FLT3) receptor has been extensively studied over the past two decades with regard to oncogenic alterations that do not only serve as prognostic markers but also as therapeutic targets in acute myeloid leukemia (AML). Internal tandem duplications (ITDs) became of special interest in this setting as they are associated with unfavorable prognosis. Because of sequence-dependent protein conformational changes FLT3-ITD tends to autophosphorylate and displays a constitutive intracellular localization. Here, we analyzed the effect of tyrosine kinase inhibitors (TKIs) on the localization of the FLT3 receptor and its mutants. TKI treatment increased the surface expression through upregulation of FLT3 and glycosylation of FLT3-ITD and FLT3-D835Y mutants. In T cell-mediated cytotoxicity (TCMC) assays, using a bispecific FLT3 × CD3 antibody construct, the combination with TKI treatment increased TCMC in the FLT3-ITD-positive AML cell lines MOLM-13 and MV4-11, patient-derived xenograft cells and primary patient samples. Our findings provide the basis for rational combination of TKI and FLT3-directed immunotherapy with potential benefit for FLT3-ITD-positive AML patients.

The Asian Rice Gall Midge (Orseolia oryzae) Mitogenome Has Evolved Novel Gene Boundaries and Tandem Repeats That Distinguish Its Biotypes.

The complete mitochondrial genome of the Asian rice gall midge, Orseolia oryzae (Diptera; Cecidomyiidae) was sequenced, annotated and analysed in the present study. The circular genome is 15,286 bp with 13 protein-coding genes, 22 tRNAs and 2 ribosomal RNA genes, and a 578 bp non-coding control region. All protein coding genes used conventional start codons and terminated with a complete stop codon. The genome presented many unusual features: (1) rearrangement in the order of tRNAs as well as protein coding genes; (2) truncation and unusual secondary structures of tRNAs; (3) presence of two different repeat elements in separate non-coding regions; (4) presence of one pseudo-tRNA gene; (5) inversion of the rRNA genes; (6) higher percentage of non-coding regions when compared with other insect mitogenomes. Rearrangements of the tRNAs and protein coding genes are explained on the basis of tandem duplication and random loss model and why intramitochondrial recombination is a better model for explaining rearrangements in the O. oryzae mitochondrial genome is discussed. Furthermore, we evaluated the number of iterations of the tandem repeat elements found in the mitogenome. This led to the identification of genetic markers capable of differentiating rice gall midge biotypes and the two Orseolia species investigated.

The strength of synergistic interaction between posaconazole and caspofungin depends on the underlying azole resistance mechanism of Aspergillus fumigatus.

The majority of azole resistance mechanisms in Aspergillus fumigatus correspond to mutations in the cyp51A gene. As azoles are less effective against infections caused by multiply azole-resistant A. fumigatus isolates, new therapeutic options are warranted for treating these infections. We therefore investigated the in vitro combination of posaconazole (POSA) and caspofungin (CAS) against 20 wild-type and resistant A. fumigatus isolates with 10 different resistance mechanisms. Fungal growth was assessed with the XTT [2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt] method. Pharmacodynamic interactions were assessed with the fractional inhibitory concentration (FIC) index (FICi) on the basis of 10% (FICi-0), 25% (FICi-1), or 53 0% (FICi-2) growth, and FICs were correlated with POSA and CAS concentrations. Synergy and antagonism were concluded when the FICi values were statistically significantly (t test, P < 0.05) lower than 1 and higher than 1.25, respectively. Significant synergy was found for all isolates with mean FICi-0 values ranging from 0.28 to 0.75 (median, 0.46). Stronger synergistic interactions were found with FICi-1 (median, 0.18; range, 0.07 to 0.47) and FICi-2 (0.31; 0.07 to 0.6). The FICi-2 values of isolates with tandem-repeat-containing mutations or codon M220 were lower than those seen with the other isolates (P < 0.01). FIC-2 values were inversely correlated with POSA MICs (rs = -0.52, P = 0.0006) and linearly with the ratio of drug concentrations in combination over the MIC of POSA (rs = 0.76, P < 0.0001) and CAS (rs = 0.52, P = 0.0004). The synergistic effect of the combination of POSA and CAS (POSA/CAS) against A. fumigatus isolates depended on the underlying azole resistance mechanism. Moreover, the drug combination synergy was found to be increased against isolates with elevated POSA MICs compared to wild-type isolates.

Inhibition of the receptor tyrosine kinase Axl impedes activation of the FLT3 internal tandem duplication in human acute myeloid leukemia: implications for Axl as a potential therapeutic target.

Approximately 20% to 25% of patients with acute myeloid leukemia (AML) have a constitutively activated FLT3-internal tandem duplication (FLT3-ITD), and these patients exhibit a poor prognosis. Here, we report that Axl, a receptor tyrosine kinase (RTK) overexpressed and constitutively active in human AML, targets the RTK FLT3 in FLT3-ITD(+) AML. Abrogation of Axl activation by soluble Axl chimeric protein (Axl-Fc) or small interfering RNA (siRNA) diminishes constitutive FLT3 phosphorylation in FLT3-ITD(+) AML. In addition, inhibition of Axl activation by Axl-Fc interferes with the physical interaction between Axl and FLT3. We found that Axl-Fc, a pharmacologic Axl inhibitor, or siRNA targeting Axl inhibits cell growth, induces cell-cycle arrest and apoptosis, and relieves a block in myeloid differentiation of FLT3-ITD(+) AML in vitro. Axl-Fc also suppresses the growth of human FLT3-ITD(+) AML in vivo. Collectively, our data suggest that Axl contributes to the pathogenesis of FLT3-ITD(+) AML through, at least in part, positive regulation of constitutive FLT3 activation. This also suggests that Axl should be pursued as a potential target for the treatment of FLT3-ITD(+) AML.

The interaction of cisplatin with a human telomeric DNA sequence containing seventeen tandem repeats.

The anti-tumour drug, cisplatin, preferentially forms adducts at G-rich DNA sequences. Telomeres are found at the ends of chromosomes and, in humans, contain the repeated DNA sequence (GGGTTA)(n) that is expected to be targeted by cisplatin. Using a plasmid clone with 17 tandem telomeric repeats, (GGGTTA)(17), the DNA sequence specificity of cisplatin was investigated utilising the linear amplification procedure that pin-pointed the precise sites of cisplatin adduct formation. This procedure used a fluorescently labelled primer and capillary electrophoresis with laser-induced fluorescence detection to determine the DNA sequence specificity of cisplatin. This technique provided a very accurate analysis of cisplatin-DNA adduct formation in a long telomeric repeat DNA sequence. The DNA sequence specificity of cisplatin in a long telomeric tandem repeat has not been previously reported. The results indicated that the 3'-end of the G-rich strand of the telomeric repeat was preferentially damaged by cisplatin and this suggests that the telomeric DNA repeat has an unusual conformation.

Mutations of FLT3/ITD confer resistance to multiple tyrosine kinase inhibitors.

FMS-like tyrosine kinase 3 (FLT3) normally functions in the survival/proliferation of hematopoietic stem/progenitor cells, but its constitutive activation by internal tandem duplication (ITD) mutations correlates with a poor prognosis in AML. The development of FLT3 tyrosine kinase inhibitors (TKI) is a promising strategy, but resistance that arises during the course of treatment caused by secondary mutations within the mutated gene itself poses a significant challenge. In an effort to predict FLT3 resistance mutations that might develop in patients, we used saturation mutagenesis of FLT3/ITD followed by selection of transfected cells in FLT3 TKI. We identified F621L, A627P, F691L and Y842C mutations in FLT3/ITD that confer varying levels of resistance to FLT3 TKI. Western blotting confirmed that some FLT3 TKI were ineffective at inhibiting FLT3 autophosphorylation and signaling through MAP kinase, STAT5 and AKT in some mutants. Balb/c mice transplanted with the FLT3/ITD Y842C mutation confirmed resistance to sorafenib in vivo but not to lestaurtinib. These results indicate a growing number of FLT3 mutations that are likely to be encountered in patients. Such knowledge, combined with known remaining sensitivity to other FLT3 TKI, will be important to establish as secondary drug treatments that can be substituted when these mutants are encountered.

MR22388, a novel anti-cancer agent with a strong FLT-3 ITD kinase affinity.

This work describes the study of the mechanism of action and spectrum of activity of MR22388, a novel anti-cancer agent belonging to the tripentone series. MR22388 is highly cytotoxic (within the nanomolar range) against numerous cancer cell lines and studies of its cytotoxicity mechanisms show that it is a weak inhibitor of the polymerization of tubulin and that it induces apoptosis via the MAP kinase pathways. Further MR22388 is a very strong inhibitor of several kinases including the tyrosine kinase FLT3-ITD. FLT3-ITD is a mutated form of the tyrosine kinase receptor (RTK) FLT3, resulting in the constitutive activation of the kinase, occurring in about 25% of normal karyotypes' Acute Myeloid Leukemia (AML) and is linked to a bad prognosis. Consecutively, MR22388 appears as a novel promising anticancer lead agent especially for AML therapy.

The DNA sequence specificity of bleomycin cleavage in telomeric sequences in human cells.

Bleomycin is an antibiotic drug that is widely used in cancer chemotherapy. Telomeres are located at the ends of chromosomes and comprise the tandemly repeated DNA sequence (GGGTTA)( n ) in humans. Since bleomycin cleaves DNA at 5'-GT dinucleotide sequences, telomeres are expected to be a major target for bleomycin cleavage. In this work, we determined the DNA sequence specificity of bleomycin cleavage in telomeric sequences in human cells. This was accomplished using a linear amplification procedure, a fluorescently labelled oligonucleotide primer and capillary gel electrophoresis with laser-induced fluorescence detection. This represents the first occasion that the DNA sequence specificity of bleomycin cleavage in telomeric DNA sequences in human cells has been reported. The bleomycin DNA sequence selectivity was mainly at 5'-GT dinucleotides, with lesser amounts at 5'-GG dinucleotides. The cellular bleomycin telomeric DNA damage was also compared with bleomycin telomeric damage in purified human genomic DNA and was found to be very similar. The implications of these results for the understanding of bleomycin's mechanism of action in human cells are discussed.

Exposure to anticancer drugs can result in transgenerational genomic instability in mice.

The genetic effects of human exposure to anticancer drugs remain poorly understood. To establish whether exposure to anticancer drugs can result not only in mutation induction in the germ line of treated animals, but also in altered mutation rates in their offspring, we evaluated mutation rates in the offspring of male mice treated with three commonly used chemotherapeutic agents: cyclophosphamide, mitomycin C, and procarbazine. The doses of paternal exposure were approximately equivalent to those used clinically. Using single-molecule PCR, the frequency of mutation at the mouse expanded simple tandem repeat locus Ms6-hm was established in DNA samples extracted from sperm and bone marrow of the offspring of treated males. After paternal exposure to any one of these three drugs, expanded simple tandem repeat mutation frequencies were significantly elevated in the germ line (sperm) and bone marrow of their offspring. This observed transgenerational instability was attributed to elevated mutation rates at the alleles derived from both the exposed fathers and from the nonexposed mothers, thus implying a genome-wide destabilization. Our results suggest that paternal exposure to a wide variety of mutagens can result in transgenerational instability manifesting in their offspring. Our data also raise important issues concerning delayed transgenerational effects in the children of survivors of anticancer therapy.

TS gene tandem repeats in esophageal cancer patients receiving chemoradiotherapy.

5-Fluorouracil (5-FU) interferes with tumor-cell proliferation by inhibiting thymidylate synthase (TS). We examined the relationship between tandem repeat (TR) variations in the TS gene and survival following concurrent chemoradiotherapy in patients with esophageal squamous cell carcinoma (ESCC). TS-TR variations were analyzed in 57 stage II-IV ESCC patients undergoing chemoradiotherapy combined with 5-FU and cisplatinum (CDDP), and in 106 controls. Pretreatment non-neoplastic biopsy specimens from ESCC patients and lymphocytes from controls were used for analysis. Variations were identified by the size of DNA fragments amplified by polymerase chain reaction. Two to five TRs were found in Japanese individuals. TR3 homozygotes were predominant in 74% of ESCC patients and 61% of controls. Three-year survival rates were significantly longer in patients with TR2/2 or TR2/3 genotypes (38%) than in patients with TR3/3, 3/4, or 3/5 genotypes (9%; p=0.011). In the Cox proportional hazard model, the TR2/2 or TR2/3 genotypes were the only independent predictor for survival (Hazard ratio, 2.647; 95% confidence interval, 1.271-5.513). The TS-TR variations exert an important influence on survival following chemoradiotherapy in ESCC patients.

Exposure to residential indoor air induces heritable DNA mutations in mice.

Exposure to the mixed indoor air pollutants released from synthetic materials and chemical products poses a serious public health problem, but little evidence has been provided to clarify whether such pollutants at environmentally relevant concentrations produce inheritable germline mutations. In the present study, mice were exposed to samples of indoor air from a newly decorated apartment bedroom. Results showed expanded simple tandem repeat (ESTR) mutations occurring in the germline of control and exposed parents, which were also detected in their offspring using three probes, Ms6-hm, Hm-2, and MMS10. Data indicated that mice being exposed to indoor air triggered a significant increase in frequency of ESTR mutations, which may be due primarily to a rise in mutations inherited through the paternal germline. These results suggest that exposure to a mixture of pollutants in indoor air obtained from an apartment in China induced ESTR mutations. Thus, humans exposed to polluted indoor apartment air in China may be at risk for developing germline mutations.

Methyl methacrylate activates the Gsta1 promoter.

Residual monomers in resin-based biomaterials cause cytotoxicity. We previously showed that methyl methacrylate (MMA) induced mRNA expression of the glutathione S-transferase alpha 1 gene (Gsta1) located downstream of the cis-acting anti-oxidant responsive element (ARE). Herein, we tested the hypothesis that MMA activated the Gsta1 promoter through the ARE. HepG2 cells were transfected with a luciferase reporter vector containing the ARE and the Gsta1 promoter (-990 to +46 bp) and cultured for 12 hrs with MMA (initial concentration, 10 mM). Analysis of the expressed luciferase activity indicated that MMA activated the promoter 2.6-fold. MMA (from 1 to 30 mM) dose-dependently increased the promoter activity, which reached a plateau between 6 and 12 hrs. In HepG2 cells transfected with a reporter vector containing 2 AREs and a TATA-like promoter, 10 mM MMA increased the reporter expression 2.8-fold. These results suggest that MMA increases Gsta1 transcription through ARE-mediated promoter activation.

The Pal3 promoter sequence is critical for the regulation of human renin gene transcription by peroxisome proliferator-activated receptor-gamma.

We recently reported that human renin gene transcription is stimulated by the nuclear receptor peroxisome proliferator-activated receptor (PPAR)-gamma in the renin-producing cell line Calu-6. The effect of PPARgamma was mapped to two sequences in the renin promoter: a direct repeat hormone response element (HRE), which is related to the classical PPAR response element (PPRE) and a nonconsensus palindromic element with a 3-bp spacer (Pal3). We now find that PPARgamma binds to the renin HRE. Neither the human renin HRE nor the consensus PPRE was sufficient to attain the maximal stimulation of renin promoter activity by the PPARgamma agonist rosiglitazone. In contrast, the human renin Pal3 element mediates both the full PPARgamma-dependent activation of transcription and the PPARgamma-driven basal renin gene transcription. The human renin Pal3 sequence was found to selectively bind PPARgamma and the retinoid X receptor-alpha from Calu-6 nuclear extracts. This is in contrast to the consensus PPRE, which can bind other nuclear proteins. PPARgamma knockdown paradoxically did not attenuate the stimulation of the endogenous renin gene expression by rosiglitazone. Similarly, a deficiency of PPARgamma did not attenuate the activation of the minimal human renin promoter, which contains the endogenous Pal3 motif. However, when the human renin Pal3 site was replaced by the consensus PPRE sequence, PPARgamma knockdown abrogated the effect of rosiglitazone on renin promoter activity. Thus, the human renin Pal3 site appears to be critical for the PPARgamma-dependent regulation of gene expression by mediating maximal transcription activation, particularly at the low cellular level of PPARgamma.

PKC 412 small-molecule tyrosine kinase inhibitor: single-compound therapy for pancreatic cancer.

BACKGROUND: PKC412 is a kinase inhibitor that blocks protein kinase C (PKC), vascular endothelial growth factor receptors, platelet-derived growth factor receptor FLT3, and other class III receptor tyrosine kinases. The enthusiasm for this compound is based on its inhibitory effect even in the case of FLT3 mutations. The aim of this study was to analyze the role of FLT3 in pancreatic cancer and to study the biological activity of combined inhibition of neovascularization and mitogenesis in this disease. METHODS: FLT3 expression was analyzed in 18 pancreatic cancer specimens by real-time quantitative polymerase chain reaction (RTQ-PCR) and immunohistochemistry. Sixteen pancreatic cancer cell lines were screened for ITD and D835 point mutations of the FLT3 gene. MTT assays and anchorage-independent growth assays were used to study cell growth. Flow cytometry was used for cell cycle analysis and apoptosis quantification. In vivo AsPC-1 and HPAF-II cells were used for orthotopic tumor modeling. Immunohistochemistry was used to quantify tumor angiogenesis. RESULTS: FLT3 expression is down-regulated in pancreatic cancer. Activating FLT3 mutations (ITD, D835) were not detectable in any of the pancreatic cancer cell lines. Cell growth was significantly inhibited as cell-cycle progression was reduced and programmed cell death increased. In vivo PKC412 therapy resulted in a significant inhibition of orthotopic tumor growth with abrogation of tumor angiogenesis. CONCLUSIONS: These data highlight that PKC412 may be a new compound in target therapy of inoperable pancreatic cancer patients and suggest a potential role for the combined use of broad spectrum kinase inhibitors in the management of these patients.