Evolving role of entrectinib in treatment of NTRK-positive tumors.

Targeted therapy has shown to be a very effective treatment in tumors with specific genomic drivers. Trk has proven to be one such target. Efforts to target the Trk fusion with specific inhibitors have shown remarkable responses in a tumor agnostic fashion, with responses seen even in patients with intracranial metastasis. Entrectinib is a first-generation Trk inhibitor with impressive activity in early phase trials performed in patients with NTRK fusion positive solid tumors and ROS1 positive non-small-cell lung cancers with subsequent approval for those indications. Entrectinib was also found to be effective in treatment of brain metastasis and generally well tolerated.

Lay abstract Advances in medical science has allowed us to analyze genes within cancer cells and target abnormal genes more precisely. One such target is called NTRK, which carries genetic information and has been targeted using a medication called entrectinib. This medication is also very effective in patients with cancers that has spread to the brain. This medication can be used in any type of cancer if the cancer cells possess the abnormal DNA. Some of the side effects of entrectinib include weight gain, lightheadedness, throwing up, taste changes, swelling of legs, lack of energy and so on. Based on the benefit of entrectinib seen in clinical trials the medication was approved by the US FDA for treatment of any type of cancer with the NTRK problem. We hope that this new approach to cancer treatment will result in patients having greater benefit and live longer.

TRK inhibitors in TRK fusion-positive cancers.

TRK fusions are oncogenic drivers of various adult and paediatric cancers. The first-generation TRK inhibitors, larotrectinib and entrectinib, were granted landmark, tumour-agnostic regulatory approvals for the treatment of these cancers in 2018 and 2019, respectively. Brisk and durable responses are achieved with these drugs in patients, including those with locally advanced or metastatic disease. In addition, intracranial activity has been observed with both agents in TRK fusion-positive solid tumours with brain metastases and primary brain tumours. While resistance to first-generation TRK inhibition can eventually occur, next-generation agents such as selitrectinib (BAY 2731954, LOXO-195) and repotrectinib were designed to address on-target resistance, which is mediated by emergent kinase domain mutations, such as those that result in substitutions at solvent front or gatekeeper residues. These next-generation drugs are currently available in the clinic and proof-of-concept responses have been reported. This underscores the utility of sequential TRK inhibitor use in select patients, a paradigm that parallels the use of targeted therapies in other oncogenic driver-positive cancers, such as ALK fusion-positive lung cancers. While TRK inhibitors have a favourable overall safety profile, select on-target adverse events, including weight gain, dizziness/ataxia and paraesthesias, are occasionally observed and should be monitored in the clinic. These side-effects are likely consequences of the inhibition of the TRK pathway that is involved in the development and maintenance of the nervous system.

In silico and in vitro screening of small molecule Inhibitors against SYT-SSX1 fusion protein in synovial sarcoma.

Synovial sarcoma (SS) is characterized by a tumour specific chromosomal translocation t(X;18) (p11;q11) which results in the formation of SYT-SSX1 fusion protein. This fusion protein represents a clear therapeutic target and molecules specifically targeting SYT-SSX1 fusion protein are currently not available. In this study, SYT-SSX1 fusion protein sequence was retrieved from Uniprot and 3D structure was generated using I-TASSER modeling program. A structure based computational screening approach has been employed using Glide docking software to identify potential SYT-SSX1 small molecule inhibitors that bind to the junction region of the fusion protein. The obtained inhibitors were further filtered based on the docking score and ADME/T properties. Ten best fit compounds were chosen for in vitro studies. The anti-proliferative activities of these 10 compounds were screened in Yamato, ASKA (carries SYT-SSX1 fusion protein) and other sarcoma cell lines such as A673, 143B to understand the specificity of inhibition of the chosen compounds. The in vitro activity was compared against HEK293 cell lines. The compound 5-fluoro-3-(1-phenyl-1H-tetraazol-5-yl)-1H-indole (FPTI) was found to be selectively cytotoxic in synovial sarcoma cell lines (Yamato and ASKA) and this compound also showed insignificant anti proliferative activity on other cell lines. Further, target gene expression study confirmed that FPTI treatment down-regulated SYT-SSX1 and modulated its downstream target genes. Cell cycle analysis revealed the involvement of an apoptotic mechanism of cell death. Further experimental validations may elucidate the therapeutic potentials of FPTI against SYT-SSX1 fusion protein.

New targets bring hope in squamous cell lung cancer: neurotrophic tyrosine kinase gene fusions.

Neurotrophic tyrosine kinase genes encode for the Trk-family proteins TrkA, TrkB, and TrkC, which have an important role in the development of the nervous system; however, they have been identified as oncogenic fusions in solid tumors (NTK-1, NTRK-2, and NTRK-3) and are associated with poor survival in lung cancer. These three new fusions can be detected by fluorescent in situ hybridization or next-generation sequencing in less than 5% of the lung tumors. There are several ongoing clinical trials of NTRK oncogenes in lung cancer and other tumors. The agents entrectinib (RXDX-101), a multi-kinase small molecule inhibitor that selectively inhibits NTRK1, NTRK2, and NTRK3, ROS1 and ALK, and LOXO-101, an ATP-competitive pan-NTRK inhibitor, have shown responses in patients with lung cancer with an acceptable toxicity profile. Although these oncogenic fusions are not very prevalent, the high prevalence of lung cancer makes these findings very relevant and suggests the feasibility of these oncogenes as targets in lung cancer. New data from Ozono and collaborators presented in this issue suggest that BDNF/TrkB signal promotes proliferating migratory and invasive phenotypes and cellular plasticity in squamous cell carcinoma (SCC) of the lung but that it also represents a druggable target that may bring hope to squamous lung cancer patients.

Combinatorial Drug Screening Identifies Ewing Sarcoma-specific Sensitivities.

Improvements in survival for Ewing sarcoma pediatric and adolescent patients have been modest over the past 20 years. Combinations of anticancer agents endure as an option to overcome resistance to single treatments caused by compensatory pathways. Moreover, combinations are thought to lessen any associated adverse side effects through reduced dosing, which is particularly important in childhood tumors. Using a parallel phenotypic combinatorial screening approach of cells derived from three pediatric tumor types, we identified Ewing sarcoma-specific interactions of a diverse set of targeted agents including approved drugs. We were able to retrieve highly synergistic drug combinations specific for Ewing sarcoma and identified signaling processes important for Ewing sarcoma cell proliferation determined by EWS-FLI1 We generated a molecular target profile of PKC412, a multikinase inhibitor with strong synergistic propensity in Ewing sarcoma, revealing its targets in critical Ewing sarcoma signaling routes. Using a multilevel experimental approach including quantitative phosphoproteomics, we analyzed the molecular rationale behind the disease-specific synergistic effect of simultaneous application of PKC412 and IGF1R inhibitors. The mechanism of the drug synergy between these inhibitors is different from the sum of the mechanisms of the single agents. The combination effectively inhibited pathway crosstalk and averted feedback loop repression, in EWS-FLI1-dependent manner. Mol Cancer Ther; 16(1); 88-101. ©2016 AACR.

High-throughput small molecule screen identifies inhibitors of aberrant chromatin accessibility.

Mutations in chromatin-modifying proteins and transcription factors are commonly associated with a wide variety of cancers. Through gain- or loss-of-function, these mutations may result in characteristic alterations of accessible chromatin, indicative of shifts in the landscape of regulatory elements genome-wide. The identification of compounds that reverse a specific chromatin signature could lead to chemical probes or potential therapies. To explore whether chromatin accessibility could serve as a platform for small molecule screening, we adapted formaldehyde-assisted isolation of regulatory elements (FAIRE), a chemical method to enrich for nucleosome-depleted genomic regions, as a high-throughput, automated assay. After demonstrating the validity and robustness of this approach, we applied this method to screen an epigenetically targeted small molecule library by evaluating regions of aberrant nucleosome depletion mediated by EWSR1-FLI1, the chimeric transcription factor critical for the bone and soft tissue tumor Ewing sarcoma. As a class, histone deacetylase inhibitors were greatly overrepresented among active compounds. These compounds resulted in diminished accessibility at targeted sites by disrupting transcription of EWSR1-FLI1. Capitalizing on precise differences in chromatin accessibility for drug discovery efforts offers significant advantages because it does not depend on the a priori selection of a single molecular target and may detect novel biologically relevant pathways.

Potential Therapeutic Approaches for the Treatment of Acute Myeloid Leukemia with AML1-ETO Translocation.

BACKGROUND: Twenty percent of patients with Acute Myeloid Leukemia (AML) carry a translocation between chromosomes 21 and chromosome 8 resulting in the formation of a chimeric oncoprotein AML1-ETO. The patients with this translocation although have a favourable prognosis, but the 5-year survival is only about 50%. It is anticipated that identification of novel therapeutic targets in t(8;21) positive AML will lead to treatment options that improve patient survival. AREAS COVERED: The oncoprotein and the proteins required to maintain its stability and functionality are the first obvious therapeutic targets. Further, newer technologies like combining gene expression and DNA occupancy profiling assays, gene expression-based high-throughput screening, etc have led to identification of proteins or pathways that are required by AML1-ETO for leukemogenesis and the agents that modulate these proteins to be considered good candidates for targeted molecular therapy. Various FDA approved drugs and secondary metabolites derived from traditional medicinal plants have been shown to possess anti-proliferative effect on t(8:21) harboring leukemic cell lines. CONCLUSION: In order to improve the therapeutic regime for AML patients with t(8;21), efforts are required to translate the success achieved in identification of potent candidates for targeted therapy into clinical setup in the best possible combination.

Pharmacologic inhibition of the Menin-MLL interaction blocks progression of MLL leukemia in vivo.

Chromosomal translocations affecting mixed lineage leukemia gene (MLL) result in acute leukemias resistant to therapy. The leukemogenic activity of MLL fusion proteins is dependent on their interaction with menin, providing basis for therapeutic intervention. Here we report the development of highly potent and orally bioavailable small-molecule inhibitors of the menin-MLL interaction, MI-463 and MI-503, and show their profound effects in MLL leukemia cells and substantial survival benefit in mouse models of MLL leukemia. Finally, we demonstrate the efficacy of these compounds in primary samples derived from MLL leukemia patients. Overall, we demonstrate that pharmacologic inhibition of the menin-MLL interaction represents an effective treatment for MLL leukemias in vivo and provide advanced molecular scaffold for clinical lead identification.

In the war against solid tumors arsenic trioxide needs partners.

In the past decade, the therapeutic potential of arsenic trioxide (ATO) in the treatment of acute promyelocytic leukemia (APL) was recognized. This encouraged other investigators to test the efficacy of ATO in the management of other hematological and solid tumor malignancies. Notably, as a single agent, arsenic trioxide did not benefit patients diagnosed with solid tumors. However, when it was combined with other agents, treatment benefit emerged. In this article, we have summarized the outcome of clinical trials that used arsenic trioxide as a single agent as well as in combination settings in patients diagnosed with solid tumors. We have also reviewed possible additional mechanisms by which ATO may be useful as a chemosensitizer in combination therapy. We hope that our review will encourage clinical investigators to rationally combine ATO with additional chemotherapeutic agents in treating patients diagnosed with solid tumors.

Targeted therapy by combined inhibition of the RAF and mTOR kinases in malignant spindle cell neoplasm harboring the KIAA1549-BRAF fusion protein.

BACKGROUND: Oncologic patients who are extreme responders to molecularly targeted therapy provide an important opportunity to better understand the biologic basis of response and, in turn, inform clinical decision making. Malignant neoplasms with an uncertain histologic and immunohistochemical characterization present challenges both on initial diagnostic workups and then later in management, as current treatment algorithms are based on a morphologic diagnosis. Herein, we report a case of a difficult to characterize sarcoma-like lesion for which genomic profiling with clinical next generation sequencing (NGS) identified the molecular underpinnings of arrested progression(stable disease) under combination targeted therapy within a phase I clinical trial. METHODS: Genomic profiling with clinical next generation sequencing was performed on the FoundationOne™ platform (Foundation Medicine, Cambridge MA). Histopathology and immunohistochemical studies were performed in the Department of Pathology, MD Anderson Cancer Center (Houston, TX). Treatment was administered in the context of a phase I clinical trial ClinicalTrials.gov Identifier: (NCT01187199). RESULTS: The histology of the tumor was that of a spindle cell neoplasm, grade 2 by FNCLCC standards. Immunohistochemical staining was positive for S100 and CD34. Genomic profiling identified the following alterations: a KIAA1549-BRAF gene fusion resulting from a tandem duplication event, a homozygous deletion of PTEN, and frameshift insertion/deletions in CDKN2A A68fs*51, SUFU E283fs*3, and MAP3K1 N325fs*3. The patient had a 25% reduction in tumor (RECIST v1.1) following combination therapy consisting of sorafenib, temsirolimus, and bevazicumab within a phase I clinical trial. CONCLUSIONS: The patient responded to combination targeted therapy that fortuitously targeted KIAA1549-BRAF and PTEN loss within a spindle cell neoplasm, as revealed by genomic profiling based on NGS. This is the first report of a tumor driven by a KIAA1549-BRAF fusion responding to sorafenib-based combination therapy.

CYT387, a selective JAK1/JAK2 inhibitor: in vitro assessment of kinase selectivity and preclinical studies using cell lines and primary cells from polycythemia vera patients.

Somatic mutations in Janus kinase 2 (JAK2), including JAK2V617F, result in dysregulated JAK-signal transducer and activator transcription (STAT) signaling, which is implicated in myeloproliferative neoplasm (MPN) pathogenesis. CYT387 is an ATP-competitive small molecule that potently inhibits JAK1/JAK2 kinases (IC(50)=11 and 18 nM, respectively), with significantly less activity against other kinases, including JAK3 (IC(50)=155 nM). CYT387 inhibits growth of Ba/F3-JAK2V617F and human erythroleukemia (HEL) cells (IC(50) approximately 1500 nM) or Ba/F3-MPLW515L cells (IC(50)=200 nM), but has considerably less activity against BCR-ABL harboring K562 cells (IC=58 000 nM). Cell lines harboring mutated JAK2 alleles (CHRF-288-11 or Ba/F3-TEL-JAK2) were inhibited more potently than the corresponding pair harboring mutated JAK3 alleles (CMK or Ba/F3-TEL-JAK3), and STAT-5 phosphorylation was inhibited in HEL cells with an IC(50)=400 nM. Furthermore, CYT387 selectively suppressed the in vitro growth of erythroid colonies harboring JAK2V617F from polycythemia vera (PV) patients, an effect that was attenuated by exogenous erythropoietin. Overall, our data indicate that the JAK1/JAK2 selective inhibitor CYT387 has potential for efficacious treatment of MPN harboring mutated JAK2 and MPL alleles.

Oridonin, a diterpenoid extracted from medicinal herbs, targets AML1-ETO fusion protein and shows potent antitumor activity with low adverse effects on t(8;21) leukemia in vitro and in vivo.

Studies have documented the potential antitumor activities of oridonin, a compound extracted from medicinal herbs. However, whether oridonin can be used in the selected setting of hematology/oncology remains obscure. Here, we reported that oridonin induced apoptosis of t(8;21) acute myeloid leukemic (AML) cells. Intriguingly, the t(8;21) product AML1-ETO (AE) fusion protein, which plays a critical role in leukemogenesis, was degraded with generation of a catabolic fragment, while the expression pattern of AE target genes investigated could be reprogrammed. The ectopic expression of AE enhanced the apoptotic effect of oridonin in U937 cells. Preincubation with caspase inhibitors blocked oridonin-triggered cleavage of AE, while substitution of Ala for Asp at residues 188 in ETO moiety of the fusion abrogated AE degradation. Furthermore, oridonin prolonged lifespan of C57 mice bearing truncated AE-expressing leukemic cells without suppression of bone marrow or reduction of body weight of animals, and exerted synergic effects while combined with cytosine arabinoside. Oridonin also inhibited tumor growth in nude mice inoculated with t(8;21)-harboring Kasumi-1 cells. These results suggest that oridonin may be a potential antileukemia agent that targets AE oncoprotein at residue D188 with low adverse effect, and may be helpful for the treatment of patients with t(8;21) AML.

Sorafenib is a potent inhibitor of FIP1L1-PDGFRalpha and the imatinib-resistant FIP1L1-PDGFRalpha T674I mutant.

The FIP1L1-PDGFRA oncogene is a common cause of chronic eosinophilic leukemia (CEL), and encodes an activated tyrosine kinase that is inhibited by imatinib. FIP1L1-PDGFRA-positive patients with CEL respond to low-dose imatinib therapy, but resistance due to acquired T674I mutation has been observed. We report here the identification of sorafenib as a potent inhibitor of the FIP1 like 1-platelet-derived growth factor receptor alpha (FIP1L1-PDGFRalpha) (T674I) mutant. Sorafenib inhibited the proliferation of FIP1L1-PDGFRalpha and FIP1L1-PDGFRalpha(T674I)-transformed Ba/F3 cells and induced apoptosis of the EOL-1 cell line at a low nanomolar concentration. Western blot analysis confirmed that these effects were due to a direct effect on FIP1L1-PDGFRalpha and FIP1L1-PDGFRalpha(T674I). Sorafenib was recently approved for the treatment of renal cell carcinoma. Our data suggest that low doses of sorafenib could be efficient for the treatment of FIP1L1-PDGFRA-positive CEL and could be used to overcome resistance to imatinib associated with the T674I mutation.

Arsenic trioxide therapy for relapsed acute promyelocytic leukemia: an useful salvage therapy.

Arsenic trioxide (As2O3) was recently identified as a very potent agent against acute promyelocytic leukemia (APL). Intravenous infusion of 10 mg As2O3 daily for one to two months can induce significant complete remission (CR) of APL, and there is no cross drug-resistance between As2O3 and other antileukemic agents, including all-trans retinoic acid (ATRA). The CR rate of relapsed and/or refractory APL patients who received As2O3 treatment ranged from 52.3% to 93.3%. The median duration to CR ranged from 38 to 51 days, with accumulative As2O3 dosage of 340-430 mg. Although most adverse reactions of As2O3 treatment were tolerable, certain infrequent but severe toxicities related to As2O3 were observed, including renal failure, hepatic damage, cardiac arrhythmia and chronic neuromuscular degeneration, which should be monitored carefully. As2O3 can induce partial differentiation and subsequent apoptosis of APL cells through degradation of wild type PML and PML/RAR alpha chimeric proteins and possible anti-mitochondrial effects. Like the treatment of ATRA in APL, early relapses from As2O3 treatment within a few months were not infrequently seen, indicating that rapid emerging resistance to As2O3 can occur. Nevertheless, the PML/RAR alpha fusion protein was reported to disappear in some APL patients who received As2O3, and who might earn long-survival. However, the follow-up is still too short to draw the conclusion. Intriguingly, it has been shown that As2O3 can also induce apoptosis of other non-APL tumor cells with clinical achievable concentrations. However, the detailed molecular mechanisms are not yet fully understood. Further studies regarding to the pharmacological characters, clinical efficacies, toxicities, apoptogenic mechanisms, and spectrum of anti-tumor activity of As2O3 are warranted.