Efficacy and safety of avapritinib in previously treated patients with advanced systemic mastocytosis.

Advanced systemic mastocytosis (AdvSM) is a rare myeloid neoplasm, driven by the KIT D816V mutation in >90% of patients. Avapritinib, a potent, highly selective D816V-mutant KIT inhibitor, is approved for treatment of adults with AdvSM by the US Food and Drug Administration, regardless of prior therapy, and the European Medicines Agency for patients with prior systemic therapy, based on EXPLORER (#NCT02561988; clinicaltrials.gov) and PATHFINDER (#NCT03580655; clinicaltrials.gov) clinical studies. We present latest pooled efficacy and safety analyses from patients who received ≥1 systemic therapy prior to avapritinib in EXPLORER/PATHFINDER. Overall response rate in response-evaluable patients (n = 31) was 71% (95% confidence interval: 52% to 86%; 22/31), including 19% (6/31) with complete remission (CR)/CR with partial recovery of peripheral blood counts (CRh). Median time to response was 2.3 months, median time to CR/CRh was 7.4 months, and median duration of response (DOR) was not reached. Reductions ≥50% in bone marrow mast cell infiltration (89%), KIT D816V variant allele fraction (66%), serum tryptase (89%), and reductions ≥35% in spleen size (70%) occurred in most patients. Median OS was not reached (median follow-up 17.7 months). Avapritinib was effective in all AdvSM subtypes, regardless of number/type of prior therapies or poor prognostic somatic mutations. Treatment-related adverse events (TRAEs) were observed in 94% of patients, most commonly grade 1/2; 57% had TRAEs of at least grade 3; 81% remained on treatment at 6 months. Avapritinib in adults with AdvSM who received prior systemic therapy was generally well tolerated, with high response rates regardless of prior systemic therapy.

The tetraamine chelator outperforms HYNIC in a new technetium-99m-labelled somatostatin receptor 2 antagonist.

BACKGROUND: Somatostatin receptor targeting radiopeptides are successfully being used to image, stage, and monitor patients with neuroendocrine tumours. They are exclusively agonists that internalise upon binding to the relevant receptor. According to recent reports, antagonists may be preferable to agonists. To date, 99mTc-labelled somatostatin receptor antagonists have attracted little attention. Here, we report on a new somatostatin receptor subtype 2 (sst2) antagonist, SS-01 (p-Cl-Phe-cyclo(D-Cys-Tyr-D-Trp-Lys-Thr-Cys)D-Tyr-NH2), with the aim of developing 99mTc-labelled ligands for SPECT/CT imaging. SS-01 was prepared using Fmoc solid-phase synthesis and subsequently coupled to the chelators 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 6-carboxy-1,4,8,11-tetraazaundecane (N4), and 6-hydrazinonicotinic acid (HYNIC) to form the corresponding peptide-chelator conjugates SS-03, SS-04, and SS-05, respectively. SS-04 and SS-05 were radiolabelled with 99mTc and SS-03 with 177Lu. Binding affinity and antagonistic properties were determined using autoradiography and immunofluorescence microscopy. Biodistribution and small animal SPECT/CT studies were performed on mice bearing HEK293-rsst2 xenografts. RESULTS: The conjugates showed low nanomolar sst2 affinity and antagonistic properties. 177Lu-DOTA-SS-01 (177Lu-SS-03) and 99mTc-N4-SS-01 (99mTc-SS-04) demonstrated high cell binding and low internalisation, whereas 99mTc-HYNIC/edda-SS-01 (99mTc-SS-05) showed practically no cellular uptake in vitro. The 99mTc-SS-04 demonstrated impressive tumour uptake at early time points, with 47% injected activity per gram tumour (%IA/g) at 1 h post-injection. The tumour uptake persisted after 4 h and was 32.5 %IA/g at 24 h. The uptake in all other organs decreased much more rapidly leading to high tumour-to-normal organ ratios, which was reflected in high-contrast SPECT/CT images. CONCLUSIONS: These data indicate a very promising 99mTc-labelled sst2-targeting antagonist. The results demonstrate high sensitivity of the 99mTc-labelling strategy, which was shown to strongly influence the receptor affinity, contrary to corresponding agonists. 99mTc-SS-04 exhibits excellent pharmacokinetics and imaging properties and appears to be a suitable candidate for SPECT/CT clinical translation.

The somatostatin receptor 2 antagonist 64Cu-NODAGA-JR11 outperforms 64Cu-DOTA-TATE in a mouse xenograft model.

Copper-64 is an attractive radionuclide for PET imaging and is frequently used in clinical applications. The aim of this study was to perform a side-by-side comparison of the in vitro and in vivo performance of 64Cu-NODAGA-JR11 (NODAGA = 1,4,7-triazacyclononane,1-glutaric acid,4,7-acetic acid, JR11 = p-Cl-Phe-cyclo(D-Cys-Aph(Hor)-D-Aph(cbm)-Lys-Thr-Cys)D-Tyr-NH2), a somatostatin receptor 2 antagonist, with the clinically used sst2 agonist 64Cu-DOTA-TATE ((TATE = D-Phe-cyclo(Cys-Tyr-D-Trp-Lys-Thr-Cys)Thr). In vitro studies demonstrated Kd values of 5.7±0.95 nM (Bmax = 4.1±0.18 nM) for the antagonist 64/natCu-NODAGA-JR11 and 20.1±4.4. nM (Bmax = 0.48±0.18 nM) for the agonist 64/natCu-DOTA-TATE. Cell uptake studies showed the expected differences between agonists and antagonists. Whereas 64Cu-DOTA-TATE (the agonist) showed very effective internalization in the cell culture assay (with 50% internalized at 4 hours post-peptide addition under the given experimental conditions), 64Cu-NODAGA-JR11 (the antagonist) showed little internalization but strong receptor-mediated uptake at the cell membrane. Biodistribution studies of 64Cu-NODAGA-JR11 showed rapid blood clearance and tumor uptake with increasing tumor-to-relevant organ ratios within the first 4 hours and in some cases, 24 hours, respectively. The tumor washout was slow or non-existent in the first 4 hours, whereas the kidney washout was very efficient, leading to high and increasing tumor-to-kidney ratios over time. Specificity of tumor uptake was proven by co-injection of high excess of non-radiolabeled peptide, which led to >80% tumor blocking. 64Cu-DOTA-TATE showed less favorable pharmacokinetics, with the exception of lower kidney uptake. Blood clearance was distinctly slower and persistent higher blood values were found at 24 hours. Uptake in the liver and lung was relatively high and also persistent. The tumor uptake was specific and similar to that of 64Cu-NODAGA-JR11 at 1 h, but release from the tumor was very fast, particularly between 4 and 24 hours. Tumor-to-normal organ ratios were distinctly lower after 1 hour. This is indicative of insufficient in vivo stability. PET studies of 64Cu-NODAGA-JR11 reflected the biodistribution data with nicely delineated tumor and low background. 64Cu-NODAGA-JR11 shows promising pharmacokinetic properties for further translation into the clinic.

Radioiodinated Exendin-4 Is Superior to the Radiometal-Labelled Glucagon-Like Peptide-1 Receptor Probes Overcoming Their High Kidney Uptake.

GLP-1 receptors are ideal targets for preoperative imaging of benign insulinoma and for quantifying the beta cell mass. The existing clinical tracers targeting GLP-1R are all agonists with low specific activity and very high kidney uptake. In order to solve those issues we evaluated GLP-1R agonist Ex-4 and antagonist Ex(9-39) radioiodinated at Tyr40 side by side with [Nle14,Lys40(Ahx-DOTA-68Ga)NH2]Ex-4 (68Ga-Ex-4) used in the clinic. The Kd, Bmax, internalization and binding kinetics of [Nle14,125I-Tyr40-NH2]Ex-4 and [Nle14,125I-Tyr40-NH2]Ex(9-39) were studied in vitro using Ins-1E cells. Biodistribution and imaging studies were performed in nude mice bearing Ins-1E xenografts. In vitro evaluation demonstrated high affinity binding of the [Nle14,125I-Tyr40-NH2]Ex-4 agonist to the Ins-1E cells with fast internalization kinetics reaching a plateau after 30 min. The antagonist [Nle14,125I-Tyr40-NH2]Ex(9-39) did not internalize and had a 4-fold higher Kd value compared to the agonist. In contrast to [Nle14,125I-Tyr40-NH2]Ex(9-39), which showed low and transient tumor uptake, [Nle14,125I-Tyr40-NH2]Ex-4 demonstrated excellent in vivo binding properties with tumor uptake identical to that of 68Ga-Ex-4, but substantially lower kidney uptake resulting in a tumor-to-kidney ratio of 9.7 at 1 h compared to 0.3 with 68Ga-Ex-4. Accumulation of activity in thyroid and stomach for both peptides, which was effectively blocked by irenat, confirms that in vivo deiodination is the mechanism behind the low kidney retention of iodinated peptides. The 124I congener of [Nle14,125I-Tyr40-NH2]Ex-4 demonstrated a similar favourable biodistribution profile in the PET imaging studies in contrast to the typical biodistribution pattern of [Nle14,Lys40(Ahx-DOTA-68Ga)NH2]Ex-4. Our results demonstrate that iodinated Ex-4 is a very promising tracer for imaging of benign insulinomas. It solves the problem of high kidney uptake of the radiometal-labelled tracers by improving the tumor-to-kidney ratio measured for [Nle14,Lys40(Ahx-DOTA-68Ga)NH2]Ex-4 by 32 fold.

Approaches to Improve the Pharmacokinetics of Radiolabeled Glucagon-Like Peptide-1 Receptor Ligands Using Antagonistic Tracers.

UNLABELLED: The glucagon-like peptide-1 (GLP-1) receptors are important biomarkers for imaging pancreatic ß-cell mass and detection of benign insulinomas. Using GLP-1 receptor antagonists, we aimed to eliminate the insulin-related side effects reported for all GLP-1 receptor agonists. Additionally, using a nonresidualizing tracer, (125)I-Bolton-Hunter-Exendin(9-39)NH2 ((125)I-BH-Ex(9-39)NH2), we aimed to reduce the high kidney uptake, enabling a better detection of insulinomas in the tail and head of the pancreas. METHODS: The affinity and biodistribution of Ex(9-39)NH2-based antagonists, modified with DOTA or NODAGA chelators at positions Lys(27) and Lys(40) and labeled with (68)Ga and (125)I-BH-Ex(9-39)NH2, were compared with the reference GLP-1 receptor agonist [Nle(14),Lys(40)(Ahx-DOTA-(68)Ga)NH2]Ex-4. The inhibitory concentration of 50% (IC50) values were determined using autoradiography on human tissues with (125)I-GLP-1(7-36)NH2 as a radioligand. Pharmacokinetics and PET imaging were studied in nude mice bearing rat Ins-1E tumors. RESULTS: Conjugation of DOTA and NODAGA chelators at positions Lys(27) and Lys(40) of Ex(9-39)NH2 resulted in a distinct loss of affinity toward GLP-1 receptor in vitro. Among the studied antagonists, [Lys(40)(NODAGA-(nat)Ga)NH2]Ex(9-39) showed the lowest IC50 value (46.7 ± 16.3 nM). The reference agonist [Nle(14),Lys(40)(Ahx-DOTA)NH2]Ex-4 demonstrated the highest affinity (IC50 = 0.9 ± 0.3 nM). Biodistribution of [Nle(14),Lys(40)(Ahx-DOTA-(68)Ga)NH2]Ex-4 at 1 h after injection demonstrated 40.2 ± 8.2 percentage injected activity per gram (%IA/g) uptake in Ins-1E tumor, 12.5 ± 2.2 %IA/g in the pancreas, and 235.8 ± 17.0 %IA/g in the kidney, with tumor-to-blood and tumor-to-kidney ratios of 100.52 and 0.17, respectively. Biodistribution of [Lys(40)(NODAGA-(68)Ga)NH2]Ex(9-39) showed only 2.2 ± 0.2 %IA/g uptake in Ins-1E tumor, 1.0 ± 0.1 %IA/g in the pancreas, and 78.4 ± 8.5 %IA/g in the kidney at 1 h after injection, with tumor-to-blood and tumor-to-kidney ratios of 7.33 and 0.03, respectively. In contrast, (125)I-BH-Ex(9-39)NH2 showed tumor uptake (42.5 ± 8.1 %IA/g) comparable to the agonist and 28.8 ± 5.1 %IA/g in the pancreas at 1 h after injection. As we hypothesized, the kidney uptake of (125)I-BH-Ex(9-39)NH2 was low, only 12.1 ± 1.4 %IA/g at 1 h after injection. The tumor-to-kidney ratio of (125)I-BH-Ex(9-39)NH2 was improved 20-fold. CONCLUSION: Our results suggest that iodinated Ex(9-39)NH2 may be a promising tracer for imaging GLP-1 receptor expression in vivo. Because of the 20-fold improved tumor-to-kidney ratio (125)I-BH-Ex(9-39)NH2 may offer higher sensitivity in the detection of insulinomas and imaging of ß-cell mass in diabetic patients. Further studies with (124)I-BH-Ex(9-39)NH2 are warranted.

Immuno-PET Imaging of CD30-Positive Lymphoma Using 89Zr-Desferrioxamine-Labeled CD30-Specific AC-10 Antibody.

UNLABELLED: The CD30-specific antibody-drug conjugate, brentuximab vedotin, is approved for the treatment of relapsed, refractory Hodgkin lymphomas and systemic anaplastic large T-cell lymphomas. Multiple ongoing clinical trials are investigating brentuximab vedotin efficacy in other CD30-positive hematologic malignancies. Because CD30 expression varies among different types of lymphoma and can also change during the course of treatment, companion diagnostic imaging of CD30 could be a valuable tool in optimizing patient-specific brentuximab vedotin treatment regimens. METHODS: The mouse antihuman CD30 antibody AC-10 was radiolabeled with the positron-emitting radionuclide (89)Zr. The stability and specificity of (89)Zr-desferrioxamine (DFO)-labeled CD30-specific AC-10 antibody ((89)Zr-DFO-AC-10) was evaluated in vitro. The pharmacokinetics of (89)Zr-DFO-AC-10 was studied in BALB/c nude mice bearing subcutaneous human Karpas 299 tumors (CD30-positive model) or A-431 tumors (CD30-negative model) using PET/CT imaging, biodistribution studies, and autoradiography. RESULTS: AC-10 was conjugated with a DFO B chelator and radiolabeled with (89)Zr to give formulated (89)Zr-DFO-AC-10 with a radiochemical yield of 80%, radiochemical purity greater than 99%, and specific activity of 111-148 MBq/mg. (89)Zr-DFO-AC-10 was stable in mouse and human sera and preserved the immunoreactivity toward CD30. Biodistribution data showed the highest tissue accumulation of (89)Zr-DFO-AC-10 in CD30-positive tumors, with 37.9% ± 8.2% injected activity per gram of tissue at 72 h after injection, whereas uptake in CD30-negative tumors was 11.0% ± 0.4%. The specificity of (89)Zr-DFO-AC-10 binding to CD30 in vivo was confirmed by blocking studies. Time-activity curves showed that between 24 and 144 h after injection, tumor-to-muscle ratios increased from 18.9 to 51.8 in the CD30-positive model and from 4.8 to 8.7 in the CD30-negative model. Tumor-to-blood ratios also increased, from 3.2 to 13.6 and from 1 to 2 in the CD30-positive and -negative models, respectively. CONCLUSION: Our results demonstrate that for measuring CD30 expression, (89)Zr-DFO-AC-10 is a sensitive PET agent with high tumor-to-normal-tissue contrast. (89)Zr-DFO-AC-10 is a promising CD30-imaging radiotracer for clinical translation in patients with various lymphomas and other diseases.

Does imaging αvß3 integrin expression with PET detect changes in angiogenesis during bevacizumab therapy?

UNLABELLED: In recent years, there has been a growing interest in molecular imaging markers of tumor-induced angiogenesis. Several radiolabeled RGD (arginine, glycine, aspartate) peptides have been developed for PET imaging of αvß3 integrins in the tumor vasculature, but there are only limited data on how angiogenesis inhibitors affect the tumor uptake of these peptides. METHODS: Changes in (68)Ga-NODAGA-c(RGDfK) peptide uptake were measured using PET during bevacizumab therapy of 2 αvß3-negative squamous cell carcinoma cell lines (A-431 and FaDu) that induce αvß3-positive neovasculature when transplanted into nude mice. Tumor uptake of (68)Ga-NODAGA-c(RGDfK) was correlated to microvascular density, vascular morphology, and permeability as well as αvß3 integrin expression. RESULTS: Bevacizumab significantly inhibited growth of A-431 tumors and caused a significant reduction in microvascular density and αvß3 integrin expression within 7 d after start of therapy. Bevacizumab also caused a normalization of blood vessel morphology and decreased tumor necrosis. However, (68)Ga-NODAGA-c(RGDfK) uptake was significantly increased at day 7 of therapy and did not decrease until after 3 wk of treatment. In Fadu xenografts, bevacizumab therapy caused only a minor inhibition of tumor growth and minor changes in (68)Ga-NODAGA-c(RGDfK) uptake. CONCLUSION: Uptake of radiolabeled RGD peptides is not necessarily decreased by effective antiangiogenic therapy. Early in the course of therapy a decrease in the expression of αvß3 integrins may not be reflected by a decrease in the uptake of RGD peptides.

In vitro differentiation of mouse embryonic stem cells into primitive blood vessels.

Mouse embryonic stem (ES) cells, derived from the inner cell mass of blastocyst stage embryos, undergo programmed differentiation in vitro to form a primitive vasculature. This programmed differentiation proceeds through similar processes of vasculogenesis and angiogenesis found during early vascular development in vivo. Partially differentiated ES cell clumps or embryoid bodies (EBs) first form blood islands that are subsequently transformed into a network of primitive blood vessels that contain lumens. Therefore, vascular differentiation of ES cells is an ideal model to study and manipulate early vascular development. Here we provide protocols for the routine maintenance of mouse ES cells and in vitro differentiation. We also include protocols for establishing transgenic ES cell lines and visualization of blood vessels by use of endothelial specific molecular markers.

Expression of deoxynucleoside kinases and 5'-nucleotidases in mouse tissues: implications for mitochondrial toxicity.

Anti-HIV nucleoside therapy can result in mitochondrial toxicity affecting muscles, peripheral nerves, pancreas and adipose tissue. The cytosolic deoxycytidine kinase (dCK; EC 2.7.1.74) and thymidine kinase (TK1; EC 2.7.1.21), the mitochondrial thymidine kinase (TK2) and deoxyguanosine kinase (dGK; EC 2.7.1.113) as well as 5'-deoxynucleotidases (5'-dNT; EC 3.1.3.5) are enzymes that control rate-limiting steps in formation of intracellular and intra-mitochondrial nucleotides. The mRNA levels and activities of these enzymes were determined in mouse tissues, using real-time PCR and selective enzyme assays. The expression of mRNA for all these enzymes and the mitochondrial deoxynucleotide carrier was detected in all tissues with a 5-10-fold variation. TK1 activities were only clearly detected in spleen and testis, while TK2, dGK and dCK activities were found in all tissues. dGK activities were higher than any other dNK in all tissues, except spleen and testis. In skeletal muscle dGK activity was 5-fold lower, TK2 and dCK levels were 10-fold lower as compared with other tissues. The variation in 5'-dNT activities was about eight-fold with the highest levels in brain and lowest in brown fat. Thus, the salvage of deoxynucleosides in muscles is 5-10-fold lower as compared to other non-proliferating tissues and 100-fold lower compared to spleen. These results may help to explain tissue specific toxicity observed with nucleoside analogs used in HIV treatment as well as symptoms in inherited mitochondrial TK2 deficiencies.

Activity profiles of deoxynucleoside kinases and 5'-nucleotidases in cultured adipocytes and myoblastic cells: insights into mitochondrial toxicity of nucleoside analogs.

Nucleoside reverse transcriptase inhibitor (NRTI) treatment of HIV is associated with complications, including lipodystrophy (LD) and myopathy. Inhibition of mitochondrial DNA polymerase and depletion of mtDNA by NRTI triphosphates are believed to be key mechanisms in NRTI toxicity. Here, we determined the activities and mRNA levels of deoxynucleoside kinases (dNK) and 5'-nucleotidases (5'-NT) controlling the rate-limiting step in intracellular phosphorylation of NRTIs in cell models representing adipose, muscle tissue and peripheral blood cells using specific assays and Taqman RT-PCR. In vitro phosphorylation of 3'-azido-2',3'-dideoxythymidine (AZT) and 2',3'-didehydro-2',3'-dideoxythymidine (d4T) in extracts was also determined. 3T3-L1 adipocytes showed similar activity of mitochondrial thymidine kinase-2 (TK2) and deoxyguanosine kinase (dGK) but 3- to 36-fold lower levels of cytosolic deoxycytidine kinase (dCK), thymidine kinase-1 (TK1) and thymidine monophosphate kinase (TMPK) and higher levels of deoxyribonucleotidase activity compared to proliferating 3T3-L1. dCK, dGK and TK2 activities correlated with their mRNA levels in proliferating, resting and differentiating 3T3-L1. Differentiated L6 myoblasts had lower activities of cytosolic dNK's and TMPK, higher dGK and similar TK2 and deoxyribonucleotidases (dNT) activities compared to proliferating myoblasts. TK2 was the limiting dNK activity while dGK was predominant in adipocytes and myocytes. Activity profiles revealed limited capacity to phosphorylate dThd and dCyd in adipocytes and myocytes compared to proliferating cells and CEM lymphocytes. Phosphorylation of AZT and d4T was low in adipocytes and myocytes, and the presence of these analogs inhibited the phosphorylation of dThd by TK2 suggesting that mitochondrial toxicity of some NRTIs in adipocytes and myocytes is due to the depletion of normal mitochondrial dNTP pools.

Flupirtine blocks apoptosis in batten patient lymphoblasts and in human postmitotic CLN3- and CLN2-deficient neurons.

Multiple gene defects cause Batten disease. Accelerated apoptosis accounts for neurodegeneration in the late infantile and juvenile forms that are due to defects in the CLN3 and CLN2 genes. Extensive neuronal death is seen in CLN2- and CLN3-deficient human brain as well as in CLN6-deficient sheep brain and retina. When neurons in late infantile and juvenile brain survive, they manage to do so by upregulating the neuroprotective molecule Bcl-2. The CLN3 gene has antiapoptotic properties at the molecular level. We show that the CLN2 gene is neuroprotective: it enhances growth of NT2 cells and maintains survival of human postmitotic hNT neurons. Conversely, blocking CLN3 or CLN2 expression in hNT neurons with adenoviral antisense-CLN3 or antisense-CLN2-AAV2 constructs causes apoptosis. The drug flupirtine is a triaminopyridine derivative that acts as a nonopioid analgesic. Flupirtine upregulates Bcl-2, increases glutathione levels, activates an inwardly rectifying potassium channel, and delays loss of intermitochondrial membrane calcium retention capacity. We show that flupirtine aborts etoposide-induced apoptosis in CLN1-, CLN2-, CLN3-, and CLN6-deficient as well as normal lymphoblasts. Flupirtine also prevents the death of CLN3- and CLN2-deficient postmitotic hNT neurons at the mitochondrial level. We show that a mechanism of neuroprotection exerted by flupirtine involves complete functional antagonism of N-methyl-D-aspartate or N-methyl-D-aspartate-induced neuronal apoptosis. Flupirtine may be useful as a drug capable of halting the progression of neurodegenerative diseases caused by dysregulated apoptosis.

The CLN3 gene is a novel molecular target for cancer drug discovery.

Juvenile Batten disease is a neurodegenerative disease caused by accelerated apoptotic death of photoreceptors and neurons attributable to defects in the CLN3 gene. CLN3 is antiapoptotic when overexpressed in NT2 neuronal precursor cells. CLN3 negatively modulates endogenous ceramide levels in NT2 cells and acts upstream of ceramide generation. Because defects in regulation of apoptosis are involved in the development of cancer, we evaluated the expression of CLN3 on both mRNA and protein levels in a variety of cancer cell lines and solid colon cancer tissue. We also observed the effect of the blocking of CLN3 protein expression on cancer cell growth, survival, ceramide production, and apoptosis by using an adenovirus-bearing antisense CLN3 construct. We show that CLN3 mRNA and protein are overexpressed in glioblastoma (U-373G and T98g), neuroblastoma (IMR-32 and SK-N-MC), prostate (Du145, PC-3, and LNCaP), ovarian (SK-OV-3, SW626, and PA-1), breast (BT-20, BT-549, and BT-474), and colon (SW1116, SW480, and HCT 116) cancer cell lines but not in pancreatic (CAPAN and As-PC-1) or lung (A-549 and NCI-H520) cancer cell lines. CLN3 is also up-regulated in mouse melanoma and breast carcinoma cancer cell lines. We found CLN3 expression is 22-330% higher than in corresponding normal colon control tissue in 8 of 10 solid colon tumors. An adenovirus-expressing antisense CLN3 (Ad-AS-CLN3) blocks CLN3 protein expression in DU-145, BT-20, SW1116, and T98g cancer cell lines as seen by Western blot. Blocking of CLN3 expression using Ad-AS-CLN3 inhibits growth and viability of cancer cells. It also causes elevation in endogenous ceramide production through de novo ceramide synthesis and results in increased apoptosis as shown by propidium iodide and JC-1 staining. This suggests that Ad-AS-CLN3 may be an option for therapy in some cancers. More importantly these results suggest that CLN3 is a novel molecular target for cancer drug discovery.