Antitumor Activity of Tasurgratinib as an Orally Available FGFR1-3 Inhibitor in Cholangiocarcinoma Models With FGFR2-fusion.

BACKGROUND/AIM: Cholangiocarcinoma (CCA) is an aggressive tumor with limited treatment options especially in 2nd line or later treatments. Targeting fibroblast growth factor receptor (FGFR) 2 has recently emerged as a promising treatment option for patients with CCA harboring FGFR2-fusion. This study investigated the antitumor activities of tasurgratinib as an orally available FGFR1-3 inhibitor, in preclinical FGFR2-driven CCA models. MATERIALS AND METHODS: Antitumor activities of tasurgratinib were examined in vitro and in vivo using NIH/3T3 cells expressing FGFR2-fusion as FGFR2-driven CCA models, and in vivo using a CCA patient-derived xenograft model. The molecular mechanism of action of tasurgratinib was elucidated through co-crystal structure analysis with FGFR1, manual complex model analysis with FGFR2, and binding kinetics analysis with FGFR2. Furthermore, the cell-based inhibitory activities against acquired resistant FGFR2 mutations in patients with CCA treated with FGFR inhibitors were evaluated. RESULTS: Tasurgratinib showed antitumor activity in preclinical FGFR2-driven CCA models by inhibiting the FGFR signaling pathway in vitro and in vivo. Furthermore, cell-based target engagement assays indicated that tasurgratinib had potent inhibitory activities against FGFR2 mutations, such as N549H/K, which are the major acquired mutations in CCA. We also confirmed that tasurgratinib exhibited fast association and slow dissociation kinetics with FGFR2, binding to the ATP-binding site and the neighboring region, and adopting an Asp-Phe-Gly (DFG)-"in" conformation. CONCLUSION: These data demonstrate the therapeutic potential of tasurgratinib in FGFR2-driven CCA and provide molecular mechanistic insights into its unique inhibitory profile against secondary FGFR2 resistance mutations in patients with CCA treated with FGFR inhibitors.

Emerging therapies in Ewing sarcoma.

PURPOSE OF REVIEW: There is an unmet need to improve outcomes for patients for Ewing sarcoma, a rare, aggressive sarcoma with a peak incidence in adolescents and young adults (AYA). Current therapy at diagnosis involves multiagent chemotherapy and local therapy, but despite intensification of treatment, those with metastases at diagnosis and recurrent disease have poor outcomes. RECENT FINDINGS: Improved understanding of Ewing sarcoma biology has identified novel targets with promising activity in Ewing sarcoma patients, including tyrosine kinase inhibitors that are now undergoing evaluation as combination and maintenance therapy. Other emerging therapies include those that target the EWSR1::FLI1 fusion oncoprotein, and act on DNA damage, cell cycle and apoptotic pathways. Immunotherapeutic approaches, particularly CAR-T-cell therapy directed at GD2, also hold promise. Recent collaborative clinical trials that have defined an international standard of care for patients with newly diagnosed Ewing sarcoma and novel platform studies with adaptive designs offer unique opportunities to investigate these therapies inclusive of all ages. SUMMARY: Close international collaboration between clinicians and biologists will allow us to prioritize promising emerging therapies and develop biomarkers to facilitate their incorporation into standard of care and more rapidly translate into benefit for Ewing sarcoma patients.

Novel therapeutic strategies targeting bypass pathways and mitochondrial dysfunction to combat resistance to RET inhibitors in NSCLC.

RET fusion is an oncogenic driver in 1-2 % of patients with non-small cell lung cancer (NSCLC). Although RET-positive tumors have been treated with multikinase inhibitors such as vandetanib or RET-selective inhibitors, ultimately resistance to them develops. Here we established vandetanib resistance (VR) clones from LC-2/ad cells harboring CCDC6-RET fusion and explored the molecular mechanism of the resistance. Each VR clone had a distinct phenotype, implying they had acquired resistance via different mechanisms. Consistently, whole exome-seq and RNA-seq revealed that the VR clones had unique mutational signatures and expression profiles, and shared only a few common remarkable events. AXL and IGF-1R were activated as bypass pathway in different VR clones, and sensitive to a combination of RET and AXL inhibitors or IGF-1R inhibitors, respectively. SMARCA4 loss was also found in a particular VR clone and 55 % of post-TKI lung tumor tissues, being correlated with higher sensitivity to SMARCA4/SMARCA2 dual inhibition and shorter PFS after subsequent treatments. Finally, we detected an increased number of damaged mitochondria in one VR clone, which conferred sensitivity to mitochondrial electron transfer chain inhibitors. Increased mitochondria were also observed in post-TKI biopsy specimens in 13/20 cases of NSCLC, suggesting a potential strategy targeting mitochondria to treat resistant tumors. Our data propose new promising therapeutic options to combat resistance to RET inhibitors in NSCLC.

Targeting fusion oncoproteins in childhood cancers: challenges and future opportunities for developing therapeutics.

Fusion oncoproteins are associated with childhood cancers and have proven challenging to target, aside from those that include kinases. As part of its efforts for targeting childhood cancers, the National Cancer Institute recently conducted a series on Novel Chemical Approaches for Targeting Fusion Oncoproteins. Key learnings on leading platforms and technologies that can be used to advance the development of molecular therapeutics that target fusion oncoproteins in childhood cancers are described. Recent breakthroughs in medicinal chemistry and chemical biology provide new ground and creative strategies to exploit for the development of targeted agents for improving outcomes against these recalcitrant cancers.

Concurrent inhibition of ALK and SRC kinases disrupts the ALK lung tumor cell proteome.

Precision oncology has revolutionized the treatment of ALK-positive lung cancer with targeted therapies. However, an unmet clinical need still to address is the treatment of refractory tumors that contain drug-induced resistant mutations in the driver oncogene or exhibit resistance through the activation of diverse mechanisms. In this study, we established mouse tumor-derived cell models representing the two most prevalent EML4-ALK variants in human lung adenocarcinomas and characterized their proteomic profiles to gain insights into the underlying resistance mechanisms. We showed that Eml4-Alk variant 3 confers a worse response to ALK inhibitors, suggesting its role in promoting resistance to targeted therapy. In addition, proteomic analysis of brigatinib-treated cells revealed the upregulation of SRC kinase, a protein frequently activated in cancer. Co-targeting of ALK and SRC showed remarkable inhibitory effects in both ALK-driven murine and ALK-patient-derived lung tumor cells. This combination induced cell death through a multifaceted mechanism characterized by profound perturbation of the (phospho)proteomic landscape and a synergistic suppressive effect on the mTOR pathway. Our study demonstrates that the simultaneous inhibition of ALK and SRC can potentially overcome resistance mechanisms and enhance clinical outcomes in ALK-positive lung cancer patients. ONE SENTENCE SUMMARY: Co-targeting ALK and SRC enhances ALK inhibitor response in lung cancer by affecting the proteomic profile, offering hope for overcoming resistance and improving clinical outcomes.

ALK-positive histiocytosis: a new clinicopathologic spectrum highlighting neurologic involvement and responses to ALK inhibition.

ALK-positive histiocytosis is a rare subtype of histiocytic neoplasm first described in 2008 in 3 infants with multisystemic disease involving the liver and hematopoietic system. This entity has subsequently been documented in case reports and series to occupy a wider clinicopathologic spectrum with recurrent KIF5B-ALK fusions. The full clinicopathologic and molecular spectra of ALK-positive histiocytosis remain, however, poorly characterized. Here, we describe the largest study of ALK-positive histiocytosis to date, with detailed clinicopathologic data of 39 cases, including 37 cases with confirmed ALK rearrangements. The clinical spectrum comprised distinct clinical phenotypic groups: infants with multisystemic disease with liver and hematopoietic involvement, as originally described (Group 1A: 6/39), other patients with multisystemic disease (Group 1B: 10/39), and patients with single-system disease (Group 2: 23/39). Nineteen patients of the entire cohort (49%) had neurologic involvement (7 and 12 from Groups 1B and 2, respectively). Histology included classic xanthogranuloma features in almost one-third of cases, whereas the majority displayed a more densely cellular, monomorphic appearance without lipidized histiocytes but sometimes more spindled or epithelioid morphology. Neoplastic histiocytes were positive for macrophage markers and often conferred strong expression of phosphorylated extracellular signal-regulated kinase, confirming MAPK pathway activation. KIF5B-ALK fusions were detected in 27 patients, whereas CLTC-ALK, TPM3-ALK, TFG-ALK, EML4-ALK, and DCTN1-ALK fusions were identified in single cases. Robust and durable responses were observed in 11/11 patients treated with ALK inhibition, 10 with neurologic involvement. This study presents the existing clinicopathologic and molecular landscape of ALK-positive histiocytosis and provides guidance for the clinical management of this emerging histiocytic entity.

In vivo inducible reverse genetics in patients' tumors to identify individual therapeutic targets.

High-throughput sequencing describes multiple alterations in individual tumors, but their functional relevance is often unclear. Clinic-close, individualized molecular model systems are required for functional validation and to identify therapeutic targets of high significance for each patient. Here, we establish a Cre-ERT2-loxP (causes recombination, estrogen receptor mutant T2, locus of X-over P1) based inducible RNAi- (ribonucleic acid interference) mediated gene silencing system in patient-derived xenograft (PDX) models of acute leukemias in vivo. Mimicking anti-cancer therapy in patients, gene inhibition is initiated in mice harboring orthotopic tumors. In fluorochrome guided, competitive in vivo trials, silencing of the apoptosis regulator MCL1 (myeloid cell leukemia sequence 1) correlates to pharmacological MCL1 inhibition in patients´ tumors, demonstrating the ability of the method to detect therapeutic vulnerabilities. The technique identifies a major tumor-maintaining potency of the MLL-AF4 (mixed lineage leukemia, ALL1-fused gene from chromosome 4) fusion, restricted to samples carrying the translocation. DUX4 (double homeobox 4) plays an essential role in patients' leukemias carrying the recently described DUX4-IGH (immunoglobulin heavy chain) translocation, while the downstream mediator DDIT4L (DNA-damage-inducible transcript 4 like) is identified as therapeutic vulnerability. By individualizing functional genomics in established tumors in vivo, our technique decisively complements the value chain of precision oncology. Being broadly applicable to tumors of all kinds, it will considerably reinforce personalizing anti-cancer treatment in the future.

Pharmaceutical Interference of the EWS-FLI1-driven Transcriptome By Cotargeting H3K27ac and RNA Polymerase Activity in Ewing Sarcoma.

The EWSR1-FLI1 t(11;22)(q24;q12) translocation is the hallmark genomic alteration of Ewing sarcoma, a malignancy of the bone and surrounding tissue, predominantly affecting children and adolescents. Although significant progress has been made for the treatment of localized disease, patients with metastasis or who relapse after chemotherapy have less than a 30% five-year survival rate. EWS-FLI1 is currently not clinically druggable, driving the need for more effective targeted therapies. Treatment with the H3K27 demethylase inhibitor, GSK-J4, leads to an increase in H3K27me and a decrease in H3K27ac, a significant event in Ewing sarcoma because H3K27ac associates strongly with EWS-FLI1 binding at enhancers and promoters and subsequent activity of EWS-FLI1 target genes. We were able to identify targets of EWS-FLI1 tumorigenesis directly inhibited by GSK-J4. GSK-J4 disruption of EWS-FLI1-driven transcription was toxic to Ewing sarcoma cells and slowed tumor growth in patient-derived xenografts (PDX) of Ewing sarcoma. Responses were markedly exacerbated by cotreatment with a disruptor of RNA polymerase II activity, the CDK7 inhibitor THZ1. This combination together suppressed EWS-FLI1 target genes and viability of ex vivo PDX Ewing sarcoma cells in a synergistic manner. In PDX models of Ewing Sarcoma, the combination shrank tumors. We present a new therapeutic strategy to treat Ewing sarcoma by decreasing H3K27ac at EWS-FLI1-driven transcripts, exacerbated by blocking phosphorylation of the C-terminal domain of RNA polymerase II to further hinder the EWS-FLI1-driven transcriptome.

Basaloid Squamous Cell Carcinoma of the Uterine Cervix: Report of a Case With Molecular Analysis.

There is a lack of knowledge about molecular alterations in basaloid squamous cell carcinoma (BSCC) of the uterine cervix. A 72-year-old woman with a history of previous subtotal hysterectomy and current vaginal bleeding was referred to our hospital. Initially, adenoid cystic carcinoma (ACC) was diagnosed upon cervical cytology and biopsy. Chest imaging showed multiple metastatic lesions in both lungs. The surgical specimen showed BSCC with diffuse p16 immunoreactivity and negativity for S-100, c-kit, and neuroendocrine markers. There was a focal minor ACC component, which could have explained the previous cytology and biopsy diagnosis. Next-generation sequencing with two different panels showed coexisting PIK3CA mutation and NTRK2 fusion with 10 additional variants of unknown significance (ATR, DAXX, FAM123B, JAK1, KEL, MLL2, NOTCH2, PALB2, POLD1, POLE). The MYB gene fusions were not identified. The patient received chemotherapy with TRK inhibitor larotrectinib and carboplatin, which caused shrinkage of metastatic lung nodules. This is the first report of cervical BSCC with extensive molecular workup, which detected multiple genetic events, including targetable ones, which are potentially implicated in the development of a tumor. The accumulation of data and further studies on this tumor are necessary to define its diagnostic criteria and its clinical and biological behavior.

Emerging therapies for inv(16) AML.

The core binding factor composed of CBFß and RUNX subunits plays a critical role in most hematopoietic lineages and is deregulated in acute myeloid leukemia (AML). The fusion oncogene CBFß-SMMHC expressed in AML with the chromosome inversion inv(16)(p13q22) acts as a driver oncogene in hematopoietic stem cells and induces AML. This review focuses on novel insights regarding the molecular mechanisms involved in CBFß-SMMHC-driven leukemogenesis and recent advances in therapeutic approaches to target CBFß-SMMHC in inv(16) AML.

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.

In silico modeling of PAX8-PPARγ fusion protein in thyroid carcinoma: influence of structural perturbation by fusion on ligand-binding affinity.

Paired box 8 (PAX8)-peroxisome proliferator-activated receptor γ (PPARγ) rearrangement is believed to play an important role in tumorigenesis of PAX8-PPARγ fusion protein (PPFP) thyroid carcinomas, while without establishing any standard treatment, including drugs. Although PPFP is a potential promising target for therapeutic agents, the three-dimensional (3D) structure and functions have not yet been experimentally elucidated. In this study, we aimed to construct the 3D structure of PPFP and to aid in the development of therapies that can target PPFP for thyroid carcinomas. The 3D structure of PPFP was constructed by homology modeling based on crystallographic structure data. To validate the modeled structure, we analyzed the thermal fluctuations by molecular dynamics simulations and predicted the physical properties using bioinformatic analyses. We found that the modeled structure was stable under hydrated conditions and had features indicating the actual existence of the structure. Furthermore, the binding free energies of the ligand rosiglitazone with PPARγ and PPFP were evaluated by the molecular mechanics-Poisson-Boltzmann surface area method. We found that rosiglitazone has different binding affinities for the same binding pockets of PPARγ and PPFP, and the optimal compound for PPFP can differ from that of PPARγ. This suggests the need for the development of drugs targeting PPFP that allow for the fusion, rather than focusing on the PPARγ side of PPFP and searching for the best compounds for that pocket. Our findings are expected to lead to the development of new therapies for thyroid tumors.

A retrospective study of alectinib versus ceritinib in patients with advanced non-small-cell lung cancer of anaplastic lymphoma kinase fusion in whom crizotinib treatment failed.

BACKGROUND: Crizotinib is the approved treatment for advanced non-small cell lung cancers (NSCLCs) of anaplastic lymphoma kinase (ALK) fusion. Failure of crizotinib treatment frequently involves drug intolerance or resistance. Comparison of using second-generation ALK inhibitors in this setting remains lacking. METHODS: Sixty-five ALK-positive advanced NSCLC patients receiving second-generation ALK inhibitors following treatment failure of crizotinib were retrospectively analyzed for the therapeutic efficacy. RESULTS: Forty-three (66.2%) and 22 (33.8%) patients received alectinib and ceritinib, respectively. Comparing alectinib to ceritinib treatment: the 12-month progression-free survival (PFS) rate (61.0% [95% confidence interval, 47.1 to 78.9%] vs. 54.5% [95% CI, 37.3 to 79.9%]); the hazard ratio (HR) for disease progression or death, 0.61 (95% CI, 0.31-1.17; p = 0.135). Multivariate Cox regression showed ECOG PS (0-1 vs. 2-3 HR 0.09 [95% CI, 0.02-0.33]; p < 0.001) and cause of crizotinib treatment failure (resistance vs. intolerance HR 2.75 [95% CI, 1.26-5.99]; p = 0.011) were the independent predictors for the PFS of second-generation ALK inhibitors. Treatment of alectinib, compared to ceritinib, was associated with a lower incidence of CNS progression (cause-specific HR, 0.10; 95% CI 0.01-0.78; p = 0.029) and a higher efficacy in patients whose cause of crizotinib treatment failure was intolerance (HR 0.29 [95% CI, 0.08-1.06]; p = 0.050). The most commonly noted adverse events were elevated AST/ALT in 10 (23.3%) patients treated with alectinib and diarrhea in 8 (36.4%) patients treated with ceritinib. CONCLUSION: Second-generation ALK inhibitors in crizotinib-treated patients showed a satifactory efficacy. Alectinib treatment demonstrated a CNS protection activity and a higher PFS in selected patients failing crizotinib treatment.

Targeting Pan-ETS Factors Inhibits Melanoma Progression.

The failure of once promising target-specific therapeutic strategies often arises from redundancies in gene expression pathways. Even with new melanoma treatments, many patients are not responsive or develop resistance, leading to disease progression in terms of growth and metastasis. We previously discovered that the transcription factors ETS1 and PAX3 drive melanoma growth and metastasis by promoting the expression of the MET receptor. Here, we find that there are multiple ETS family members expressed in melanoma and that these factors have redundant functions. The small molecule YK-4-279, initially developed to target the ETS gene-containing translocation product EWS-FLI1, significantly inhibited cellular growth, invasion, and ETS factor function in melanoma cell lines and a clinically relevant transgenic mouse model, BrafCA;Tyr-CreERT2;Ptenf/f. One of the antitumor effects of YK-4-279 in melanoma is achieved via interference of multiple ETS family members with PAX3 and the expression of the PAX3-ETS downstream gene MET. Expression of exogenous MET provided partial rescue of the effects of YK-4-279, further supporting that MET loss is a significant contributor to the antitumor effects of the drug. This is the first study identifying multiple overlapping functions of the ETS family promoting melanoma. In addition, targeting all factors, rather than individual members, demonstrated impactful deleterious consequences in melanoma progression. Given that multiple ETS factors are known to have oncogenic functions in other malignancies, these findings have a high therapeutic impact. SIGNIFICANCE: These findings identify YK-4-279 as a promising therapeutic agent against melanoma by targeting multiple ETS family members and blocking their ability to act as transcription factors.

Molecular Characterization and Therapeutic Targeting of Colorectal Cancers Harboring Receptor Tyrosine Kinase Fusions.

PURPOSE: Receptor tyrosine kinase fusions in colorectal cancers are rare, but potentially therapeutically relevant. We describe clinical, molecular, and pathologic attributes of RTK fusion-associated colorectal cancer. EXPERIMENTAL DESIGN: We identified all cases with RTK fusions in patients with colorectal cancer seen at Dana-Farber Cancer Institute (Boston, MA) who underwent OncoPanel testing between 2013 and 2018. Clinical, histologic, and molecular features were extracted from the patient charts and molecular testing results. RESULTS: We identified 12 driver oncogenic fusions in various RTKs. These fusions occurred exclusively in BRAF and RAS wild-type tumors and were enriched in right-sided and mismatch repair-deficient (MMR-D) colorectal cancers. All of the MMR-D colorectal cancers with RTK fusions were found in tumors with acquired MMR-D due to MLH1 promoter hypermethylation and one was associated with a sessile serrated polyp. Molecular profiles of MMR-D colorectal cancer with RTK fusions largely resembled BRAF V600E-mutated MMR-D colorectal cancer, rather than those secondary to Lynch syndrome. We describe two patients with fusion-associated microsatellite stable (MSS) colorectal cancer who derived clinical benefit from therapeutic targeting of their translocation. The first harbored an ALK-CAD fusion and received sequential crizotinib and alectinib therapy for a total of 7.5 months until developing an ALK L1196Q gatekeeper mutation. The second patient, whose tumor contained an ROS1-GOPC fusion, continues to benefit from entrectinib after 9 months of therapy. CONCLUSIONS: RTK fusions in colorectal cancer are a rare, but important disease subgroup that occurs in RAS and BRAF wild-type tumors. Despite enrichment in acquired MMR-D tumors, RTK fusions also occur in MSS colorectal cancer and provide an important therapeutic target.

Opposing immune and genetic mechanisms shape oncogenic programs in synovial sarcoma.

Synovial sarcoma (SyS) is an aggressive neoplasm driven by the SS18-SSX fusion, and is characterized by low T cell infiltration. Here, we studied the cancer-immune interplay in SyS using an integrative approach that combines single-cell RNA sequencing (scRNA-seq), spatial profiling and genetic and pharmacological perturbations. scRNA-seq of 16,872 cells from 12 human SyS tumors uncovered a malignant subpopulation that marks immune-deprived niches in situ and is predictive of poor clinical outcomes in two independent cohorts. Functional analyses revealed that this malignant cell state is controlled by the SS18-SSX fusion, is repressed by cytokines secreted by macrophages and T cells, and can be synergistically targeted with a combination of HDAC and CDK4/CDK6 inhibitors. This drug combination enhanced malignant-cell immunogenicity in SyS models, leading to induced T cell reactivity and T cell-mediated killing. Our study provides a blueprint for investigating heterogeneity in fusion-driven malignancies and demonstrates an interplay between immune evasion and oncogenic processes that can be co-targeted in SyS and potentially in other malignancies.

Fusion genes as biomarkers in pediatric cancers: A review of the current state and applicability in diagnostics and personalized therapy.

The incidence of pediatric cancers is rising steadily across the world, along with the challenges in understanding the molecular mechanisms and devising effective therapeutic strategies. Pediatric cancers are presented with diverse molecular characteristics and more distinct subtypes when compared to adult cancers. Recent studies on the genomic landscape of pediatric cancers using next-generation sequencing (NGS) approaches have redefined this field by providing better subtype characterization and novel actionable targets. Since early identification and personalized treatment strategies influence therapeutic outcomes, survival, and quality of life in pediatric cancer patients, the quest for actionable biomarkers is of great value in this field. Fusion genes that are prevalent and recurrent in several pediatric cancers are ideally suited in this context due to their disease-specific occurrence. In this review, we explore the current status of fusion genes in pediatric cancer subtypes and their use as biomarkers for diagnosis and personalized therapy. We discuss the technological advancements made in recent years in NGS sequencing and their impact on fusion detection algorithms that have revolutionized this field. Finally, we also discuss the advantages of pairing liquid biopsy protocols for fusion detection and their eventual use in diagnosis and treatment monitoring.