Whole Exome Sequencing reveals NOTCH1 mutations in anaplastic large cell lymphoma and points to Notch both as a key pathway and a potential therapeutic target.

Patients diagnosed with Anaplastic Large Cell Lymphoma (ALCL) are still treated with toxic multi-agent chemotherapy and as many as 25-50% of patients relapse. To understand disease pathology and to uncover novel targets for therapy, Whole-Exome Sequencing (WES) of Anaplastic Lymphoma Kinase (ALK)+ ALCL was performed as well as Gene-Set Enrichment Analysis. This revealed that the T-cell receptor (TCR) and Notch pathways were the most enriched in mutations. In particular, variant T349P of NOTCH1, which confers a growth advantage to cells in which it is expressed, was detected in 12% of ALK+ and ALK- ALCL patient samples. Furthermore, we demonstrate that NPM-ALK promotes NOTCH1 expression through binding of STAT3 upstream of NOTCH1. Moreover, inhibition of NOTCH1 with γ-secretase inhibitors (GSIs) or silencing by shRNA leads to apoptosis; co-treatment in vitro with the ALK inhibitor Crizotinib led to additive/synergistic anti-tumour activity suggesting this may be an appropriate combination therapy for future use in the circumvention of ALK inhibitor resistance. Indeed, Crizotinib-resistant and sensitive ALCL were equally sensitive to GSIs. In conclusion, we show a variant in the extracellular domain of NOTCH1 that provides a growth advantage to cells and confirm the suitability of the Notch pathway as a second-line druggable target in ALK+ ALCL.

Three novel patient-derived BCR/ABL mutants show different sensitivity to second and third generation tyrosine kinase inhibitors.

BCR/ABL (Breakpoint Cluster Region protein/Abelson tyrosine-protein kinase 1) kinase domain (KD) mutations represent the most frequently described mechanism of resistance to the treatment with tyrosine kinase inhibitors (TKI) in patients with chronic myeloid leukemia (CML). Mutations may impair TKI activity by directly or indirectly impairing the drug binding to the protein. We report the discovery of three new BCR/ABL mutations, L248R, T315V, and F317R identified in two patients with CML (L248R and T315V) and in one patient with Ph+ acute lymphoblastic leukemia (ALL) (F317R). Mutations were screened against second-generation (bosutinib, nilotinib, and dasatinib), as well as third-generation TKIs (ponatinib/AP-24534 and DCC-2036). Furthermore, the activity profile of ponatinib and DCC-2036 against a panel of 24 clinically relevant BCR/ABL mutants is presented and compared to the other TKIs. The IC50 values for each TKI against the mutants and the IC50 increase over wild type BCR/ABL (relative resistance, RR) were calculated to define four resistance levels: sensitive (RR ≤ 2), moderately resistant (2 < RR ≤ 4), resistant (4 < RR ≤ 10), or highly resistant (RR > 10). L248R and T315V showed high resistance to imatinib, bosutinib, dasatinib, and nilotinib, intermediate resistance to ponatinib, but were sensitive to DCC-2036. Interestingly, F317R showed a moderate resistance to imatinib and nilotinib, but is resistant/highly resistant to dasatinib, bosutinib, ponatinib, and DCC-2036. The availability of drugs activity profiles may become a useful tool for clinicians dealing with the treatment of drug-resistant CML patients.

Identification of non-ATP-competitive α-carboline inhibitors of the anaplastic lymphoma kinase.

The Anaplastic Lymphoma Kinase (ALK) is a therapeutic target for personalized medicine in selected cancers. Despite excellent clinical responses to ALK inhibitors, most patients develop drug resistance and relapse. New compounds with alternative binding modes are needed to overcome resistant mutants. Here we describe a medicinal chemistry effort to the design and development of novel ALK inhibitors based on a 4,6-substituted α-carboline scaffold. Active compounds were able to inhibit the gatekeeper L1196M mutant, in several cases better than the wild-type enzyme. Compound 43 showed potent non-ATP-competitive inhibition of wild-type and mutant ALK, including G1202R, in biochemical and cellular assays, as well as in xenograft mouse models.

Discovery of Novel α-Carboline Inhibitors of the Anaplastic Lymphoma Kinase.

The anaplastic lymphoma kinase (ALK) is abnormally expressed and hyperactivated in a number of tumors and represents an ideal therapeutic target. Despite excellent clinical responses to ALK inhibition, drug resistance still represents an issue and novel compounds that overcome drug-resistant mutants are needed. We designed, synthesized, and evaluated a large series of azacarbazole inhibitors. Several lead compounds endowed with submicromolar potency were identified. Compound 149 showed selective inhibition of native and mutant drug-refractory ALK kinase in vitro as well as in a Ba/F3 model and in human ALK+ lymphoma cells. The three-dimensional (3D) structure of a 149:ALK-KD cocrystal is reported, showing extensive interaction through the hinge region and the catalytic lysine 1150.

Correction to "Discovery of Novel α-Carboline Inhibitors of the Anaplastic Lymphoma Kinase".

[This corrects the article DOI: 10.1021/acsomega.2c00507.].

Synergistic Drug Combinations Prevent Resistance in ALK+ Anaplastic Large Cell Lymphoma.

Anaplastic lymphoma kinase-positive (ALK+) anaplastic large-cell lymphoma (ALCL) is a subtype of non-Hodgkin lymphoma characterized by expression of the oncogenic NPM/ALK fusion protein. When resistant or relapsed to front-line chemotherapy, ALK+ ALCL prognosis is very poor. In these patients, the ALK inhibitor crizotinib achieves high response rates, however 30-40% of them develop further resistance to crizotinib monotherapy, indicating that new therapeutic approaches are needed in this population. We here investigated the efficacy of upfront rational drug combinations to prevent the rise of resistant ALCL, in vitro and in vivo. Different combinations of crizotinib with CHOP chemotherapy, decitabine and trametinib, or with second-generation ALK inhibitors, were investigated. We found that in most cases combined treatments completely suppressed the emergence of resistant cells and were more effective than single drugs in the long-term control of lymphoma cells expansion, by inducing deeper inhibition of oncogenic signaling and higher rates of apoptosis. Combinations showed strong synergism in different ALK-dependent cell lines and better tumor growth inhibition in mice. We propose that drug combinations that include an ALK inhibitor should be considered for first-line treatments in ALK+ ALCL.

Mitochondrial Hyperactivation and Enhanced ROS Production are Involved in Toxicity Induced by Oncogenic Kinases Over-Signaling.

Targeted therapy is an effective, rational, and safe approach to solid and hematological tumors treatment. Unfortunately, a significant fraction of patients treated with tyrosine kinase inhibitors (TKI) relapses mainly because of gene amplification, mutations, or other bypass mechanisms. Recently a growing number of papers showed how, in some cases, resistance due to oncogene overexpression may be associated with drug addiction: cells able to proliferate in the presence of high TKI doses become also TKI dependent, undergoing cellular stress, and apoptosis/death upon drug withdrawal. Notably, if a sub-cellular population survives TKI discontinuation it is also partially re-sensitized to the same drug. Thus, it is possible that a subset of patients relapsing upon TKI treatment may benefit from a discontinuous therapeutic schedule. We focused on two different hematologic malignancies, chronic myeloid leukemia (CML) and anaplastic large cell lymphoma (ALCL), both successfully treatable with TKIs. The two models utilized (LAMA and SUP-M2) differed in having oncogene overexpression as the sole cause of drug resistance (CML), or additionally carrying kinase domain mutations (ALCL). In both cases drug withdrawal caused a sudden overload of oncogenic signal, enhanced mitochondria activity, induced the release of a high amount of reactive oxygen species (ROS), and caused genotoxic stress and massive cell death. In LAMA cells (CML) we could rescue the cells from death by partially blocking downstream oncogenic signaling or lowering ROS detrimental effect by adding reduced glutathione.

Lorlatinib Treatment Elicits Multiple On- and Off-Target Mechanisms of Resistance in ALK-Driven Cancer.

: Targeted therapy changed the standard of care in ALK-dependent tumors. However, resistance remains a major challenge. Lorlatinib is a third-generation ALK inhibitor that inhibits most ALK mutants resistant to current ALK inhibitors. In this study, we utilize lorlatinib-resistant anaplastic large cell lymphoma (ALCL), non-small cell lung cancer (NSCLC), and neuroblastoma cell lines in vitro and in vivo to investigate the acquisition of resistance and its underlying mechanisms. ALCL cells acquired compound ALK mutations G1202R/G1269A and C1156F/L1198F in vitro at high drug concentrations. ALCL xenografts selected in vivo showed recurrent N1178H (5/10 mice) and G1269A (4/10 mice) mutations. Interestingly, intracellular localization of NPM/ALKN1178H skewed toward the cytoplasm in human cells, possibly mimicking overexpression. RNA sequencing of resistant cells showed significant alteration of PI3K/AKT and RAS/MAPK pathways. Functional validation by small-molecule inhibitors confirmed the involvement of these pathways in resistance to lorlatinib. NSCLC cells exposed in vitro to lorlatinib acquired hyperactivation of EGFR, which was blocked by erlotinib to restore sensitivity to lorlatinib. In neuroblastoma, whole-exome sequencing and proteomic profiling of lorlatinib-resistant cells revealed a truncating NF1 mutation and hyperactivation of EGFR and ErbB4. These data provide an extensive characterization of resistance mechanisms that may arise in different ALK-positive cancers following lorlatinib treatment. SIGNIFICANCE: High-throughput genomic, transcriptomic, and proteomic profiling reveals various mechanisms by which multiple tumor types acquire resistance to the third-generation ALK inhibitor lorlatinib.

Synergistic activity of ALK and mTOR inhibitors for the treatment of NPM-ALK positive lymphoma.

ALK-positive Anaplastic Large Cell Lymphoma (ALCL) represents a subset of Non-Hodgkin Lymphoma whose treatment benefited from crizotinib development, a dual ALK/MET inhibitor. Crizotinib blocks ALK-triggered pathways such as PI3K/AKT/mTOR, indispensable for survival of ALK-driven tumors.Despite the positive impact of targeted treatment in ALCL, resistant clones are often selected during therapy. Strategies to overcome resistance include the design of second generation drugs and the use of combined therapies that simultaneously target multiple nodes essential for cells survival. We investigated the effects of combined ALK/mTOR inhibition. We observed a specific synergistic effect of combining ALK inhibitors with an mTOR inhibitor (temsirolimus), in ALK+ lymphoma cells. The positive cooperation resulted in an increased inhibition of mTOR effectors, compared to single treatments, a block in G0/G1 phase and induction of apoptosis. The combination was able to prevent the selection of resistant clones, while long-term exposure to single agents led to the establishment of resistant cell lines, with either ALK inhibitor or temsirolimus. In vivo, mice injected with Karpas 299 cells and treated with low dose combination showed complete regression of tumors, while only partial inhibition was obtained in single agents-treated mice. Upon treatment stop the combination was able to significantly delay tumor relapses. Re-challenge of relapsed tumors at a higher dose led to full regression of xenografts in the combination group, but not in mice treated with lorlatinib alone. In conclusion, our data suggest that the combination of ALK and mTOR inhibitors could be a valuable therapeutic option for ALK+ ALCL patients.

Excess of NPM-ALK oncogenic signaling promotes cellular apoptosis and drug dependency.

Most of the anaplastic large-cell lymphoma (ALCL) cases carry the t(2;5; p23;q35) that produces the fusion protein NPM-ALK (nucleophosmin-anaplastic lymphoma kinase). NPM-ALK-deregulated kinase activity drives several pathways that support malignant transformation of lymphoma cells. We found that in ALK-rearranged ALCL cell lines, NPM-ALK was distributed in equal amounts between the cytoplasm and the nucleus. Only the cytoplasmic portion was catalytically active in both cell lines and primary ALCL, whereas the nuclear portion was inactive because of heterodimerization with NPM1. Thus, about 50% of the NPM-ALK is not active and sequestered as NPM-ALK/NPM1 heterodimers in the nucleus. Overexpression or relocalization of NPM-ALK to the cytoplasm by NPM genetic knockout or knockdown caused ERK1/2 (extracellular signal-regulated protein kinases 1 and 2) increased phosphorylation and cell death through the engagement of an ATM/Chk2- and γH2AX (phosphorylated H2A histone family member X)-mediated DNA-damage response. Remarkably, human NPM-ALK-amplified cell lines resistant to ALK tyrosine kinase inhibitors (TKIs) underwent apoptosis upon drug withdrawal as a consequence of ERK1/2 hyperactivation. Altogether, these findings indicate that an excess of NPM-ALK activation and signaling induces apoptosis via oncogenic stress responses. A 'drug holiday' where the ALK TKI treatment is suspended could represent a therapeutic option in cells that become resistant by NPM-ALK amplification.

Activity of second-generation ALK inhibitors against crizotinib-resistant mutants in an NPM-ALK model compared to EML4-ALK.

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor involved in both solid and hematological tumors. About 80% of ALK-positive anaplastic large-cell lymphoma (ALCL) cases are characterized by the t(2;5)(p23;q35) translocation, encoding for the aberrant fusion protein nucleophosmin (NPM)-ALK, whereas 5% of non-small-cell lung cancer (NSCLC) patients carry the inv(2)(p21;p23) rearrangement, encoding for the echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion. The ALK/c-MET/ROS inhibitor crizotinib successfully improved the treatment of ALK-driven diseases. However, several cases of resistance appeared in NSCLC patients, and ALK amino acid substitutions were identified as a leading cause of resistance to crizotinib. Second-generation ALK inhibitors have been developed in order to overcome crizotinib resistance. In this work, we profiled in vitro the activity of crizotinib, AP26113, ASP3026, alectinib, and ceritinib against six mutated forms of ALK associated with clinical resistance to crizotinib (C1156Y, L1196M, L1152R, G1202R, G1269A, and S1206Y) and provide a classification of mutants according to their level of sensitivity/resistance to the drugs. Since the biological activity of ALK mutations extends beyond the specific type of fusion, both NPM-ALK- and EML4-ALK-positive cellular models were used. Our data revealed that most mutants may be targeted by using different inhibitors. One relevant exception is represented by the G1202R substitution, which was highly resistant to all drugs (>10-fold increased IC50 compared to wild type) and may represent the most challenging mutation to overcome. These results provide a prediction of cross-resistance of known crizotinib-resistant mutations against all second-generation tyrosine kinase inhibitors (TKIs) clinically available, and therefore could be a useful tool to help clinicians in the management of crizotinib-resistance cases.

NPM/ALK mutants resistant to ASP3026 display variable sensitivity to alternative ALK inhibitors but succumb to the novel compound PF-06463922.

ALK is involved in the onset of several tumors. Crizotinib (XalkoriTM), a potent ALK inhibitor, represents the current front-line treatment for ALK+ NSCLC and shows great clinical efficacy. However, resistant disease often develops after initial response. ASP3026 is a novel second-generation ALK inhibitor with activity on crizotinib-resistant ALK-L1196M gatekeeper mutant. As resistance is likely to be a relevant hurdle for any drug, we sought to determine the resistance profile of ASP3026 in the context of NPM/ALK+ ALCL. We selected six ASP3026-resistant cell lines by culturing human ALCL cells in the presence of increasing concentrations of drug. The established resistant cell lines carry several point mutations in the ALK kinase domain (G1128S, C1156F, I1171N/T, F1174I, N1178H, E1210K and C1156F/D1203N were the most frequent) that are shown to confer resistance to ASP3026 in the Ba/F3 cell model. All mutants were profiled for cross-resistance against a panel of clinically relevant inhibitors including ceritinib, alectinib, crizotinib, AP26113 and PF-06463922. Finally, a genetically heterogeneous ASP3026-resistant cell line was exposed to second-line treatment simulations with all inhibitors. The population evolved according to relative sensitivity of its mutant subclones to the various drugs. Compound PF-06463922 did not allow the outgrowth of any resistant clone, at non-toxic doses.

In vitro and in vivo identification of ABCB1 as an efflux transporter of bosutinib.

BACKGROUND: Bosutinib is a recently approved ABL inhibitor. In spite of the well-documented effectiveness of BCR-ABL inhibitors in treating chronic myeloid leukemia, development of resistance is a continuous clinical challenge. Transporters that facilitate drug uptake and efflux have been proposed as one potential source of resistance to tyrosine kinase inhibitor treatment. Our aim was to determine which carriers are responsible for bosutinib transport. METHODS: K562S cells overexpressing the drug transporters ABCB1, ABCG2, and SLC22A1 were generated, characterized and used in proliferation assay and intracellular uptake and retention assay (IUR). In vivo experiments were performed in nude mice injected with K562S, K562DOX cells (overexpressing ABCB1), and K562DOX silenced for ABCB1 (K562DOX/sh P-GP). RESULTS: The IUR assay using C-14 bosutinib showed that only ABCB1 was responsible for active bosutinib transport. K562DOX cells showed the lowest intracellular level of bosutinib, while K562DOX cells treated with the ABCB1 inhibitor verapamil showed intracellular bosutinib levels comparable with parental K562S. Proliferation assays demonstrated that K562DOX are resistant to bosutinib treatment while verapamil is able to restore the sensitivity to the drug. Nude mice injected with K562DOX and treated with bosutinib showed very limited response and quickly relapsed after stopping treatment while K562S as well as K562DOX/sh P-GP remained tumor-free. CONCLUSIONS: Our data suggest that the analysis of ABCB1 expression levels might help determine treatment options for patients exhibiting resistance to bosutinib.

Reversal of microRNA-150 silencing disadvantages crizotinib-resistant NPM-ALK(+) cell growth.

The regulatory microRNA miR-150 is involved in the development of hemopathies and is downregulated in T-lymphomas, such as anaplastic large-cell lymphoma (ALCL) tumors. ALCL is defined by the presence or absence of translocations that activate the anaplastic lymphoma kinase (ALK), with nucleophosmin-ALK (NPM-ALK) fusions being the most common. Here, we compared samples of primary NPM-ALK(+) and NPM-ALK(-) ALCL to investigate the role of miR-150 downstream of NPM-ALK. Methylation of the MIR150 gene was substantially elevated in NPM-ALK(+) biopsies and correlated with reduced miR-150 expression. In NPM-ALK(+) cell lines, DNA hypermethylation-mediated miR-150 repression required ALK-dependent pathways, as ALK inhibition restored miR-150 expression. Moreover, epigenetic silencing of miR-150 was due to the activation of STAT3, a major downstream substrate of NPM-ALK, in cooperation with DNA methyltransferase 1 (DNMT1). Accordingly, miR-150 repression was turned off following treatment with the DNMT inhibitor, decitabine. In murine NPM-ALK(+) xenograft models, miR-150 upregulation induced antineoplastic activity. Treatment of crizotinib-resistant NPM-ALK(+) KARPAS-299-CR06 cells with decitabine or ectopic miR-150 expression reduced viability and growth. Altogether, our results suggest that hypomethylating drugs, alone or in combination with other agents, may benefit ALK(+) patients harboring tumors resistant to crizotinib and other anti-ALK tyrosine kinase inhibitors (TKIs). Moreover, these results support further work on miR-150 in these and other ALK(+) malignancies.

Ceritinib as a promising therapy for ALK related diseases.

Ceritinib, also known as LDK-378 or Zykadia (Novartis), is a second generation inhibitor able to specifically target the anaplastic lymphoma kinase (ALK). In the last five years the interest for ALK small inhibitors grew rapidly, mainly because it was discovered that a small but significant percentage of non-small cell lung cancer (NSCLC) patients carries the oncogenic fusion protein EML4-ALK, in addition to about half percent of anaplastic large cell lymphoma (ALCL) patients, an aggressive but definitely rarer non Hodgkin's T cell lymphoma, and other malignancies. Moreover the first ALK inhibitor, crizotinib (Xalkori or PF02341066) was successfully approved for the treatment of late stages or metastatic ALK+ NSCLC, giving a new, safer therapeutic option for those patients. As predicted from previous clinical experience with other kinase inhibitors, crizotinib resistance inevitably occurred, so the clinical availability of new compounds able to overcome crizotinib resistance became a priority. Recently the first clinical data from the phase I trial on ceritinib were published (N Engl J Med 2014;370:1189-97): 59 patients were enrolled in the dose-escalation phase while additional 71 patients were treated in the following expansion phase. For 19 patients relapsed upon crizotinib treatment, ceritinib was used as second line therapy. Collectively, ORR was 58%, 56% for patients who received crizotinib before. Maximum tolerated dose (MTD) was established at 750 mg daily, but more than half patients had to reduce the drug dose because of adverse events. Finally PFS was 7.0 months. Here we discuss the clinical data presented in this article, comparing ceritinib with the first line inhibitor crizotinib and another second generation ALK inhibitor, alectinib (Chugai-Roche).

Crizotinib in advanced, chemoresistant anaplastic lymphoma kinase-positive lymphoma patients.

Anaplastic lymphoma kinase (ALK)-positive lymphomas respond to chemotherapy, but relapses, which bear a poor prognosis, occur. Crizotinib inhibits ALK in vitro and in vivo and was administered as monotherapy to 11 ALK+ lymphoma patients who were resistant/refractory to cytotoxic therapy. The overall response rate was 10 of 11 (90.9%; 95% confidence interval [CI] = 58.7% to 99.8%). Disease status at the latest follow-up is as follows: four patients are in complete response (CR) (months >21, >30, >35, >40) under continuous crizotinib administration; 4 patients had progression of disease (months 1, 2, 2, 2); 1 patient obtained CR on crizotinib, received an allogeneic bone marrow transplant, and is in CR; 2 patients (treated before and/or after allogeneic bone marrow transplant) obtained and are still in CR but they have stopped crizotinib. Overall and progression-free survival rates at 2 years are 72.7% (95% CI = 39.1% to 94.0%) and 63.7% (95% CI = 30.8% to 89.1%), respectively. ALK mutations conferring resistance to crizotinib in vitro could be identified in relapsed patients. Crizotinib exerted a potent antitumor activity with durable responses in advanced, heavily pretreated ALK+ lymphoma patients, with a benign safety profile.

Crizotinib-resistant NPM-ALK mutants confer differential sensitivity to unrelated Alk inhibitors.

The dual ALK/MET inhibitor crizotinib was recently approved for the treatment of metastatic and late-stage ALK+ NSCLC, and is currently in clinical trial for other ALK-related diseases. As predicted after other tyrosine kinase inhibitors' clinical experience, the first mutations that confer resistance to crizotinib have been described in patients with non-small cell lung cancer (NSCLC) and in one patient inflammatory myofibroblastic tumor (IMT). Here, we focused our attention on the anaplastic large cell lymphoma (ALCL), where the oncogenic fusion protein NPM-ALK, responsible for 70% to 80% of cases, represents an ideal crizotinib target. We selected and characterized 2 human NPM-ALK+ ALCL cell lines, KARPAS-299 and SUP-M2, able to survive and proliferate at different crizotinib concentrations. Sequencing of ALK kinase domain revealed that a single mutation became predominant at high crizotinib doses in each cell line, namely L1196Q and I1171N in Karpas-299 and SUP-M2 cells, respectively. These mutations also conferred resistance to crizotinib in Ba/F3 cells expressing human NPM-ALK. The resistant cell populations, as well as mutated Ba/F3 cells, were characterized for sensitivity to two additional ALK inhibitors: the dual ALK/EGFR inhibitor AP26113 and NVP-TAE684. While L1196Q-positive cell lines were sensitive to both inhibitors, cells carrying I1171N substitution showed cross-resistance to all ALK inhibitors tested. This study provides potentially relevant information for the management of patients with ALCL that may relapse after crizotinib treatment.

Synergistic effects of combined Wnt/KRAS inhibition in colorectal cancer cells.

Activation of Wnt signalling due to inability to degrade ß-catenin is found in >85% of colorectal cancers. Approximately half of colon cancers express a constitutively active KRAS protein. A significant fraction of patients show both abnormalities. We previously reported that simultaneous down-regulation of both ß-catenin and KRAS was necessary to induce significant cell death and tumor growth inhibition of colorectal cancer cells. Although attractive, an RNAi-based therapeutic approach is still far from being employed in the clinical setting. Therefore, we sought to recapitulate our previous findings by the use of small-molecule inhibitors of ß-catenin and KRAS. We show here that the ß-catenin inhibitors PKF115-584 and pyrvinium pamoate block ß-catenin-dependent transcriptional activity and synergize with the KRAS inhibitor S-trans, trans-farnesylthiosalicylic acid (FTS, salirasib) in colon cancer cells driven by Wnt and KRAS oncogenic signals, but not in cells carrying BRAF mutations. The combined use of these compounds was superior to the use of any drug alone in inducing cell growth arrest, cell death, MYC and survivin down-modulation, and inhibition of anchorage-independent growth. Expression analysis of selected cancer-relevant genes revealed down-regulation of CD44 as a common response to the combined treatments. These data provide a proof of principle for a combination therapeutic strategy in colorectal cancer.

Colorectal tumors are effectively eradicated by combined inhibition of {beta}-catenin, KRAS, and the oncogenic transcription factor ITF2.

Colorectal carcinomas (CRC) harbor well-defined genetic abnormalities, including aberrant activation of ß-catenin (ß-cat) and KRAS, but independent targeting of these molecules seems to have limited therapeutic effect. In this study, we report therapeutic effects of combined targeting of different oncogenes in CRC. Inducible short hairpin RNA (shRNA)-mediated silencing of ß-cat, ITF2, or KRAS decreased proliferation by 88%, 72%, and 45%, respectively, with no significant apoptosis in any case. In contrast, combined blockade of ß-cat and ITF2 inhibited proliferation by 99% with massive apoptosis. Similar effects occurred after combined shRNA against ß-cat and KRAS. In vivo, single oncogene blockade inhibited the growth of established tumors by up to 30%, whereas dual ß-cat and ITF2 targeting caused 93% inhibition. Similar tumor growth suppression was achieved by double ß-cat/KRAS shRNA in vivo. Our findings illustrate an effective therapeutic principle in CRC based on a combination targeting strategy that includes the ITF2 oncogene, which represents a novel therapeutic target.

iNKT cells control mouse spontaneous carcinoma independently of tumor-specific cytotoxic T cells.

BACKGROUND: CD1d-restricted invariant NKT (iNKT) cells are a subset of T lymphocytes endowed with innate effector functions that aid in the establishment of adaptive T and B cell immune responses. iNKT cells have been shown to play a spontaneous protective role against experimental tumors. Yet, the interplay between iNKT and tumor-specific T cells in cancer immune surveillance/editing has never been addressed. The transgenic adenocarcinoma of the mouse prostate (TRAMP) is a realistic model of spontaneous oncogenesis, in which the tumor-specific cytotoxic T cell (CTL) response undergoes full tolerance upon disease progression. PRINCIPAL FINDINGS: We report here that lack of iNKT cells in TRAMP mice resulted in the appearance of more precocious and aggressive tumors that significantly reduced animal survival. TRAMP mice bearing or lacking iNKT cells responded similarly to a tumor-specific vaccination and developed tolerance to a tumor-associated antigen at comparable rate. CONCLUSIONS: Hence, our data argue for a critical role of iNKT cells in the immune surveillance of carcinoma that is independent of tumor-specific CTL.