Monica Ceccon

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.

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.

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.

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.

Treatment Efficacy and Resistance Mechanisms Using the Second-Generation ALK Inhibitor AP26113 in Human NPM-ALK–Positive Anaplastic Large Cell Lymphoma

ALK is a tyrosine kinase receptor involved in a broad range of solid and hematologic tumors. Among 70% to 80% of ALK+ anaplastic large cell lymphomas (ALCL) are caused by the aberrant oncogenic fusion protein NPM-ALK. Crizotinib was the first clinically relevant ALK inhibitor, now approved for the treatment of late-stage and metastatic cases of lung cancer. However, patients frequently develop drug resistance to Crizotinib, mainly due to the appearance of point mutations located in the ALK kinase domain. Fortunately, other inhibitors are available and in clinical trial, suggesting the potential for second-line therapies to overcome Crizotinib resistance. This study focuses on the ongoing phase I/II trial small-molecule tyrosine kinase inhibitor (TKI) AP26113, by Ariad Pharmaceuticals, which targets both ALK and EGFR. Two NPM-ALK+ human cell lines, KARPAS-299 and SUP-M2, were grown in the presence of increasing concentrations of AP26113, and eight lines were selected that demonstrated resistance. All lines show IC50 values higher (130 to 1,000-fold) than the parental line. Mechanistically, KARPAS-299 populations resistant to AP26113 show NPM-ALK overexpression, whereas SUP-M2–resistant cells harbor several point mutations spanning the entire ALK kinase domain. In particular, amino acid substitutions: L1196M, S1206C, the double F1174V+L1198F and L1122V+L1196M mutations were identified. The knowledge of the possible appearance of new clinically relevant mechanisms of drug resistance is a useful tool for the management of new TKI-resistant cases. Implications: This work defines reliable ALCL model systems of AP26113 resistance and provides a valuable tool in the management of all cases of relapse upon NPM-ALK–targeted therapy. Mol Cancer Res; 13(4); 775–83. ©2014 AACR.

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 β-catenin (β-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 β-cat, ITF2, or KRAS decreased proliferation by 88%, 72%, and 45%, respectively, with no significant apoptosis in any case. In contrast, combined blockade of β-cat and ITF2 inhibited proliferation by 99% with massive apoptosis. Similar effects occurred after combined shRNA against β-cat and KRAS. In vivo, single oncogene blockade inhibited the growth of established tumors by up to 30%, whereas dual β-cat and ITF2 targeting caused 93% inhibition. Similar tumor growth suppression was achieved by double β-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.

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

Activation of Wnt signalling due to inability to degrade β-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 β-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 β-catenin and KRAS. We show here that the β-catenin inhibitors PKF115-584 and pyrvinium pamoate block β-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.

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

BackgroundBosutinib 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.MethodsK562S 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).ResultsThe 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.ConclusionsOur 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.

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.