Targeting TRIP13 in favorable histology Wilms tumor with nuclear export inhibitors synergizes with doxorubicin.

Wilms tumor (WT) is the most common renal malignancy of childhood. Despite improvements in the overall survival, relapse occurs in ~15% of patients with favorable histology WT (FHWT). Half of these patients will succumb to their disease. Identifying novel targeted therapies remains challenging in part due to the lack of faithful preclinical in vitro models. Here we establish twelve patient-derived WT cell lines and demonstrate that these models faithfully recapitulate WT biology using genomic and transcriptomic techniques. We then perform loss-of-function screens to identify the nuclear export gene, XPO1, as a vulnerability. We find that the FDA approved XPO1 inhibitor, KPT-330, suppresses TRIP13 expression, which is required for survival. We further identify synergy between KPT-330 and doxorubicin, a chemotherapy used in high-risk FHWT. Taken together, we identify XPO1 inhibition with KPT-330 as a potential therapeutic option to treat FHWTs and in combination with doxorubicin, leads to durable remissions in vivo.

Identification of Circulating Serum Multi-MicroRNA Signatures in Human DLBCL Models.

There remains a need to identify new sensitive diagnostic and predictive blood-based platforms in lymphoma. We previously discovered a novel circulating microRNA (miRNA) signature in a Smurf2-deficient mouse model that spontaneously develops diffuse large B-cell lymphoma (DLBCL). Herein, we investigated this 10-miRNA signature (miR-15a, let-7c, let-7b, miR-27a, miR-10b, miR-18a, miR-497, miR-130a, miR24, and miR-155) in human lymphoma cell lines, mice engrafted with patient-derived xenografts (PDXs), and DLBCL patient serum samples leveraging systems biology analyses and droplet digital PCR (ddPCR) technology. Overall, 90% of the miRNAs were enriched in PDX DLBCL models and human lymphoma cell lines. Circulating miRNAs from the serum of 86 DLBCL patients were significantly increased compared with healthy controls and had similar patterns to the murine models. Strikingly, miRNAs were identified up to 27-fold higher levels in the serum of PDX-bearing mice and human patients compared with lymphoma cell lysates, suggesting a concentration of these factors over time within sera. Using cut-points from recursive partitioning analysis, we derived a 5-miRNA signature (let-7b, let-7c, miR-18a, miR-24, and miR-15a) with a classification rate of 91% for serum from patients with DLBCL versus normal controls. In addition, higher levels of circulating let-7b miRNA were associated with more advanced stage disease (i.e., III-IV vs. I-II) in DLBCL patients and higher levels of miR-27a and miR-24 were associated with MYC rearrangement. Taken together, circulating multi-miRNAs were readily detectable in pre-clinical cell line and human lymphoma models as well as in DLBCL patients where they appeared to distinguish clinico-pathologic subtypes and disease features.

DNA methyltransferase inhibition overcomes diphthamide pathway deficiencies underlying CD123-targeted treatment resistance.

The interleukin-3 receptor α subunit, CD123, is expressed in many hematologic malignancies including acute myeloid leukemia (AML) and blastic plasmacytoid dendritic cell neoplasm (BPDCN). Tagraxofusp (SL-401) is a CD123-targeted therapy consisting of interleukin-3 fused to a truncated diphtheria toxin payload. Factors influencing response to tagraxofusp other than CD123 expression are largely unknown. We interrogated tagraxofusp resistance in patients and experimental models and found that it was not associated with CD123 loss. Rather, resistant AML and BPDCN cells frequently acquired deficiencies in the diphthamide synthesis pathway, impairing tagraxofusp's ability to ADP-ribosylate cellular targets. Expression of DPH1, encoding a diphthamide pathway enzyme, was reduced by DNA CpG methylation in resistant cells. Treatment with the DNA methyltransferase inhibitor azacitidine restored DPH1 expression and tagraxofusp sensitivity. We also developed a drug-dependent ADP-ribosylation assay in primary cells that correlated with tagraxofusp activity and may represent an additional novel biomarker. As predicted by these results and our observation that resistance also increased mitochondrial apoptotic priming, we found that the combination of tagraxofusp and azacitidine was effective in patient-derived xenografts treated in vivo. These data have important implications for clinical use of tagraxofusp and led to a phase 1 study combining tagraxofusp and azacitidine in myeloid malignancies.

Mechanisms of Lymphoma Clearance Induced by High-Dose Alkylating Agents.

The extraordinary activity of high-dose cyclophosphamide against some high-grade lymphomas was described nearly 60 years ago. Here we address mechanisms that mediate cyclophosphamide activity in bona fide human double-hit lymphoma. We show that antibody resistance within the bone marrow (BM) is not present upon early engraftment but develops during lymphoma progression. This resistance required a high tumor:macrophage ratio, was recapitulated in spleen by partial macrophage depletion, and was overcome by multiple, high-dose alkylating agents. Cyclophosphamide induced endoplasmic reticulum (ER) stress in BM-resident lymphoma cells in vivo that resulted in ATF4-mediated paracrine secretion of VEGFA, massive macrophage infiltration, and clearance of alemtuzumab-opsonized cells. BM macrophages isolated after cyclophosphamide treatment had increased phagocytic capacity that was reversed by VEGFA blockade or SYK inhibition. Single-cell RNA sequencing of these macrophages identified a "super-phagocytic" subset that expressed CD36/FCGR4. Together, these findings define a novel mechanism through which high-dose alkylating agents promote macrophage-dependent lymphoma clearance. SIGNIFICANCE: mAbs are effective against only a small subset of cancers. Herein, we recapitulate compartment-specific antibody resistance and define an ER stress-dependent mechanism induced by high-dose alkylating agents that promotes phagocytosis of opsonized tumor cells. This approach induces synergistic effects with mAbs and merits testing across additional tumor types.See related commentary by Duval and De Palma, p. 834.This article is highlighted in the In This Issue feature, p. 813.

Biomarker-driven strategy for MCL1 inhibition in T-cell lymphomas.

There is a pressing need for more effective therapies to treat patients with T-cell lymphomas (TCLs), including first-line approaches that increase the response rate to cyclophosphamide, adriamycin, vincristine, and prednisone (CHOP) chemotherapy. We characterized the mitochondrial apoptosis pathway in cell lines and patient-derived xenograft (PDX) models of TCL and assessed the in vitro efficacy of BH3 mimetics, including the BCL2 inhibitor venetoclax, the BCL2/BCL-xL inhibitor navitoclax, and the novel MCL1 inhibitor AZD5991. The abundance of antiapoptotic BCL2 family members based on immunoblotting or RNA transcript levels correlated poorly with the activity of BH3 mimetics. In contrast, the functional approach BH3 profiling reliably predicted sensitivity to BH3 mimetics in vitro and in vivo. We used BH3 profiling to select TCL PDX that were dependent on MCL1. Mice xenografted with these PDX and treated with AZD5991 had markedly improved survival. The combination of AZD5991 and CHOP achieved synergy based on survival improvement beyond a mathematical "sum of benefits" model. Thus, MCL1 inhibition is a promising strategy as both a single agent and in combination with chemotherapy for patients with TCL and functional dependence on MCL1.

Targetable vulnerabilities in T- and NK-cell lymphomas identified through preclinical models.

T- and NK-cell lymphomas (TCL) are a heterogenous group of lymphoid malignancies with poor prognosis. In contrast to B-cell and myeloid malignancies, there are few preclinical models of TCLs, which has hampered the development of effective therapeutics. Here we establish and characterize preclinical models of TCL. We identify multiple vulnerabilities that are targetable with currently available agents (e.g., inhibitors of JAK2 or IKZF1) and demonstrate proof-of-principle for biomarker-driven therapies using patient-derived xenografts (PDXs). We show that MDM2 and MDMX are targetable vulnerabilities within TP53-wild-type TCLs. ALRN-6924, a stapled peptide that blocks interactions between p53 and both MDM2 and MDMX has potent in vitro activity and superior in vivo activity across 8 different PDX models compared to the standard-of-care agent romidepsin. ALRN-6924 induced a complete remission in a patient with TP53-wild-type angioimmunoblastic T-cell lymphoma, demonstrating the potential for rapid translation of discoveries from subtype-specific preclinical models.

Pan-SRC kinase inhibition blocks B-cell receptor oncogenic signaling in non-Hodgkin lymphoma.

In diffuse large B-cell lymphoma (DLBCL), activation of the B-cell receptor (BCR) promotes multiple oncogenic signals, which are essential for tumor proliferation. Inhibition of the Bruton's tyrosine kinase (BTK), a BCR downstream target, is therapeutically effective only in a subgroup of patients with DLBCL. Here, we used lymphoma cells isolated from patients with DLBCL to measure the effects of targeted therapies on BCR signaling and to anticipate response. In lymphomas resistant to BTK inhibition, we show that blocking BTK activity enhanced tumor dependencies from alternative oncogenic signals downstream of the BCR, converging on MYC upregulation. To completely ablate the activity of the BCR, we genetically and pharmacologically repressed the activity of the SRC kinases LYN, FYN, and BLK, which are responsible for the propagation of the BCR signal. Inhibition of these kinases strongly reduced tumor growth in xenografts and cell lines derived from patients with DLBCL independent of their molecular subtype, advancing the possibility to be relevant therapeutic targets in broad and diverse groups of DLBCL patients.

PDX-MI: Minimal Information for Patient-Derived Tumor Xenograft Models.

Patient-derived tumor xenograft (PDX) mouse models have emerged as an important oncology research platform to study tumor evolution, mechanisms of drug response and resistance, and tailoring chemotherapeutic approaches for individual patients. The lack of robust standards for reporting on PDX models has hampered the ability of researchers to find relevant PDX models and associated data. Here we present the PDX models minimal information standard (PDX-MI) for reporting on the generation, quality assurance, and use of PDX models. PDX-MI defines the minimal information for describing the clinical attributes of a patient's tumor, the processes of implantation and passaging of tumors in a host mouse strain, quality assurance methods, and the use of PDX models in cancer research. Adherence to PDX-MI standards will facilitate accurate search results for oncology models and their associated data across distributed repository databases and promote reproducibility in research studies using these models. Cancer Res; 77(21); e62-66. ©2017 AACR.

A B Cell Regulome Links Notch to Downstream Oncogenic Pathways in Small B Cell Lymphomas.

Gain-of-function Notch mutations are recurrent in mature small B cell lymphomas such as mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL), but the Notch target genes that contribute to B cell oncogenesis are largely unknown. We performed integrative analysis of Notch-regulated transcripts, genomic binding of Notch transcription complexes, and genome conformation data to identify direct Notch target genes in MCL cell lines. This B cell Notch regulome is largely controlled through Notch-bound distal enhancers and includes genes involved in B cell receptor and cytokine signaling and the oncogene MYC, which sustains proliferation of Notch-dependent MCL cell lines via a Notch-regulated lineage-restricted enhancer complex. Expression of direct Notch target genes is associated with Notch activity in an MCL xenograft model and in CLL lymph node biopsies. Our findings provide key insights into the role of Notch in MCL and other B cell malignancies and have important implications for therapeutic targeting of Notch-dependent oncogenic pathways.

Characterization of midostaurin as a dual inhibitor of FLT3 and SYK and potentiation of FLT3 inhibition against FLT3-ITD-driven leukemia harboring activated SYK kinase.

Oncogenic FLT3 kinase is a clinically validated target in acute myeloid leukemia (AML), and both multi-targeted and selective FLT3 inhibitors have been developed. Spleen tyrosine kinase (SYK) has been shown to be activated and increased in FLT3-ITD-positive AML patients, and has further been shown to be critical for transformation and maintenance of the leukemic clone in these patients. Further, over-expression of constitutively activated SYK causes resistance to highly selective FLT3 tyrosine kinase inhibitors (TKI). Up to now, the activity of the multi-targeted FLT3 inhibitor, midostaurin, against cells expressing activated SYK has not been explored in the context of leukemia, although SYK has been identified as a target of midostaurin in systemic mastocytosis. We compared the ability of midostaurin to inhibit activated SYK in mutant FLT3-positive AML cells with that of inhibitors displaying dual SYK/FLT3 inhibition, targeted SYK inhibition, and targeted FLT3 inhibition. Our findings suggest that dual FLT3/SYK inhibitors and FLT3-targeted drugs potently kill oncogenic FLT3-transformed cells, while SYK-targeted small molecule inhibition displays minimal activity. However, midostaurin and other dual FLT3/SYK inhibitors display superior anti-proliferative activity when compared to targeted FLT3 inhibitors, such as crenolanib and quizartinib, against cells co-expressing FLT3-ITD and constitutively activated SYK-TEL. Interestingly, additional SYK suppression potentiated the effects of dual FLT3/SYK inhibitors and targeted FLT3 inhibitors against FLT3-ITD-driven leukemia, both in the absence and presence of activated SYK. Taken together, our findings have important implications for the design of drug combination studies in mutant FLT3-positive patients and for the design of future generations of FLT3 inhibitors.

HSP90 inhibition overcomes ibrutinib resistance in mantle cell lymphoma.

The Bruton tyrosine kinase (BTK) inhibitor ibrutinib induces responses in 70% of patients with relapsed and refractory mantle cell lymphoma (MCL). Intrinsic resistance can occur through activation of the nonclassical NF-κB pathway and acquired resistance may involve the BTK C481S mutation. Outcomes after ibrutinib failure are dismal, indicating an unmet medical need. We reasoned that newer heat shock protein 90 (HSP90) inhibitors could overcome ibrutinib resistance by targeting multiple oncogenic pathways in MCL. HSP90 inhibition induced the complete degradation of both BTK and IκB kinase α in MCL lines and CD40-dependent B cells, with downstream loss of MAPK and nonclassical NF-κB signaling. A proteome-wide analysis in MCL lines and an MCL patient-derived xenograft identified a restricted set of targets from HSP90 inhibition that were enriched for factors involved in B-cell receptor and JAK/STAT signaling, the nonclassical NF-κB pathway, cell-cycle regulation, and DNA repair. Finally, multiple HSP90 inhibitors potently killed MCL lines in vitro, and the clinical agent AUY922 was active in vivo against both patient-derived and cell-line xenografts. Together, these findings define the HSP90-dependent proteome in MCL. Considering the disappointing clinical activity of HSP90 inhibitors in other contexts, trials in patients with MCL will be essential for defining the efficacy of and mechanisms of resistance after ibrutinib failure.

The Public Repository of Xenografts Enables Discovery and Randomized Phase II-like Trials in Mice.

More than 90% of drugs with preclinical activity fail in human trials, largely due to insufficient efficacy. We hypothesized that adequately powered trials of patient-derived xenografts (PDX) in mice could efficiently define therapeutic activity across heterogeneous tumors. To address this hypothesis, we established a large, publicly available repository of well-characterized leukemia and lymphoma PDXs that undergo orthotopic engraftment, called the Public Repository of Xenografts (PRoXe). PRoXe includes all de-identified information relevant to the primary specimens and the PDXs derived from them. Using this repository, we demonstrate that large studies of acute leukemia PDXs that mimic human randomized clinical trials can characterize drug efficacy and generate transcriptional, functional, and proteomic biomarkers in both treatment-naive and relapsed/refractory disease.

Diffuse large B-cell lymphoma patient-derived xenograft models capture the molecular and biological heterogeneity of the disease.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease defined by transcriptional classifications, specific signaling and survival pathways, and multiple low-frequency genetic alterations. Preclinical model systems that capture the genetic and functional heterogeneity of DLBCL are urgently needed. Here, we generated and characterized a panel of large B-cell lymphoma (LBCL) patient-derived xenograft (PDX) models, including 8 that reflect the immunophenotypic, transcriptional, genetic, and functional heterogeneity of primary DLBCL and 1 that is a plasmablastic lymphoma. All LBCL PDX models were subjected to whole-transcriptome sequencing to classify cell of origin and consensus clustering classification (CCC) subtypes. Mutations and chromosomal rearrangements were evaluated by whole-exome sequencing with an extended bait set. Six of the 8 DLBCL models were activated B-cell (ABC)-type tumors that exhibited ABC-associated mutations such as MYD88, CD79B, CARD11, and PIM1. The remaining 2 DLBCL models were germinal B-cell type, with characteristic alterations of GNA13, CREBBP, and EZH2, and chromosomal translocations involving IgH and either BCL2 or MYC Only 25% of the DLBCL PDX models harbored inactivating TP53 mutations, whereas 75% exhibited copy number alterations of TP53 or its upstream modifier, CDKN2A, consistent with the reported incidence and type of p53 pathway alterations in primary DLBCL. By CCC criteria, 6 of 8 DLBCL PDX models were B-cell receptor (BCR)-type tumors that exhibited selective surface immunoglobulin expression and sensitivity to entospletinib, a recently developed spleen tyrosine kinase inhibitor. In summary, we have established and characterized faithful PDX models of DLBCL and demonstrated their usefulness in functional analyses of proximal BCR pathway inhibition.

Mediator kinase inhibition further activates super-enhancer-associated genes in AML.

Super-enhancers (SEs), which are composed of large clusters of enhancers densely loaded with the Mediator complex, transcription factors and chromatin regulators, drive high expression of genes implicated in cell identity and disease, such as lineage-controlling transcription factors and oncogenes. BRD4 and CDK7 are positive regulators of SE-mediated transcription. By contrast, negative regulators of SE-associated genes have not been well described. Here we show that the Mediator-associated kinases cyclin-dependent kinase 8 (CDK8) and CDK19 restrain increased activation of key SE-associated genes in acute myeloid leukaemia (AML) cells. We report that the natural product cortistatin A (CA) selectively inhibits Mediator kinases, has anti-leukaemic activity in vitro and in vivo, and disproportionately induces upregulation of SE-associated genes in CA-sensitive AML cell lines but not in CA-insensitive cell lines. In AML cells, CA upregulated SE-associated genes with tumour suppressor and lineage-controlling functions, including the transcription factors CEBPA, IRF8, IRF1 and ETV6 (refs 6-8). The BRD4 inhibitor I-BET151 downregulated these SE-associated genes, yet also has anti-leukaemic activity. Individually increasing or decreasing the expression of these transcription factors suppressed AML cell growth, providing evidence that leukaemia cells are sensitive to the dosage of SE-associated genes. Our results demonstrate that Mediator kinases can negatively regulate SE-associated gene expression in specific cell types, and can be pharmacologically targeted as a therapeutic approach to AML.

Activity of the Type II JAK2 Inhibitor CHZ868 in B Cell Acute Lymphoblastic Leukemia.

A variety of cancers depend on JAK2 signaling, including the high-risk subset of B cell acute lymphoblastic leukemias (B-ALLs) with CRLF2 rearrangements. Type I JAK2 inhibitors induce paradoxical JAK2 hyperphosphorylation in these leukemias and have limited activity. To improve the efficacy of JAK2 inhibition in B-ALL, we developed the type II inhibitor CHZ868, which stabilizes JAK2 in an inactive conformation. CHZ868 potently suppressed the growth of CRLF2-rearranged human B-ALL cells, abrogated JAK2 signaling, and improved survival in mice with human or murine B-ALL. CHZ868 and dexamethasone synergistically induced apoptosis in JAK2-dependent B-ALLs and further improved in vivo survival compared to CHZ868 alone. These data support the testing of type II JAK2 inhibition in patients with JAK2-dependent leukemias and other disorders.

Identification of novel therapeutic targets in acute leukemias with NRAS mutations using a pharmacologic approach.

Oncogenic forms of NRAS are frequently associated with hematologic malignancies and other cancers, making them important therapeutic targets. Inhibition of individual downstream effector molecules (eg, RAF kinase) have been complicated by the rapid development of resistance or activation of bypass pathways. For the purpose of identifying novel targets in NRAS-transformed cells, we performed a chemical screen using mutant NRAS transformed Ba/F3 cells to identify compounds with selective cytotoxicity. One of the compounds identified, GNF-7, potently and selectively inhibited NRAS-dependent cells in preclinical models of acute myelogenous leukemia and acute lymphoblastic leukemia. Mechanistic analysis revealed that its effects were mediated in part through combined inhibition of ACK1/AKT and of mitogen-activated protein kinase kinase kinase kinase 2 (germinal center kinase). Similar to genetic synthetic lethal approaches, these results suggest that small molecule screens can be used to identity novel therapeutic targets in cells addicted to RAS oncogenes.

Identification of Wee1 as a novel therapeutic target for mutant RAS-driven acute leukemia and other malignancies.

Direct targeting of rat sarcoma (RAS), which is frequently mutated, has proven to be challenging, and inhibition of individual downstream RAS mediators has resulted in limited clinical efficacy. We designed a chemical screen to identify compounds capable of potentiating mammalian target of rapamycin (mTOR) inhibition in mutant RAS-positive leukemia, and identified a Wee1 inhibitor. Synergy was observed in both mutant neuroblastoma RAS viral oncogene homolog (NRAS)- and mutant kirsten RAS viral oncogene homolog (KRAS)-positive acute myelogenous leukemia (AML) cell lines and primary patient samples. The observed synergy enhanced dephosphorylation of AKT, 4E-binding protein 1 and s6 kinase, and correlated with increased apoptosis. The specificity of Wee1 as the target of MK-1775 was validated by Wee1 knockdown, as well as partial reversal of drug combination-induced apoptosis by a cyclin-dependent kinase 1 (CDK1) inhibitor. Importantly, we also extended our findings to other mutant RAS-expressing malignancies, including mutant NRAS-positive melanoma, and mutant KRAS-positive colorectal cancer, pancreatic cancer and lung cancer. We observed favorable responses with combined Wee1/mTOR inhibition in human cancer cell lines from multiple malignancies, and inhibition of tumor growth in in vivo models of mutant KRAS lung cancer and leukemia. The present study introduces for the first time Wee1 inhibition combined with mTOR inhibition as a novel therapeutic strategy for the selective treatment of mutant RAS-positive leukemia and other mutant RAS-expressing malignancies.

Mutations in G protein ß subunits promote transformation and kinase inhibitor resistance.

Activating mutations in genes encoding G protein α (Gα) subunits occur in 4-5% of all human cancers, but oncogenic alterations in Gß subunits have not been defined. Here we demonstrate that recurrent mutations in the Gß proteins GNB1 and GNB2 confer cytokine-independent growth and activate canonical G protein signaling. Multiple mutations in GNB1 affect the protein interface that binds Gα subunits as well as downstream effectors and disrupt Gα interactions with the Gßγ dimer. Different mutations in Gß proteins clustered partly on the basis of lineage; for example, all 11 GNB1 K57 mutations were in myeloid neoplasms, and seven of eight GNB1 I80 mutations were in B cell neoplasms. Expression of patient-derived GNB1 variants in Cdkn2a-deficient mouse bone marrow followed by transplantation resulted in either myeloid or B cell malignancies. In vivo treatment with the dual PI3K-mTOR inhibitor BEZ235 suppressed GNB1-induced signaling and markedly increased survival. In several human tumors, mutations in the gene encoding GNB1 co-occurred with oncogenic kinase alterations, including the BCR-ABL fusion protein, the V617F substitution in JAK2 and the V600K substitution in BRAF. Coexpression of patient-derived GNB1 variants with these mutant kinases resulted in inhibitor resistance in each context. Thus, GNB1 and GNB2 alterations confer transformed and resistance phenotypes across a range of human tumors and may be targetable with inhibitors of G protein signaling.

Molecular rationale for the use of PI3K/AKT/mTOR pathway inhibitors in combination with crizotinib in ALK-mutated neuroblastoma.

Mutations in the ALK tyrosine kinase receptor gene represent important therapeutic targets in neuroblastoma, yet their clinical translation has been challenging. The ALK(F1174L) mutation is sensitive to the ALK inhibitor crizotinib only at high doses and mediates acquired resistance to crizotinib in ALK-translocated cancers. We have shown that the combination of crizotinib and an inhibitor of downstream signaling induces a favorable response in transgenic mice bearing ALK(F1174L)/MYCN-positive neuroblastoma. Here, we investigated the molecular basis of this effect and assessed whether a similar strategy would be effective in ALK-mutated tumors lacking MYCN overexpression. We show that in ALK-mutated, MYCN-amplified neuroblastoma cells, crizotinib alone does not affect mTORC1 activity as indicated by persistent RPS6 phosphorylation. Combined treatment with crizotinib and an ATP-competitive mTOR inhibitor abrogated RPS6 phosphorylation, leading to reduced tumor growth and prolonged survival in ALK(F1174L)/MYCN-positive models compared to single agent treatment. By contrast, this combination, while inducing mTORC1 downregulation, caused reciprocal upregulation of PI3K activity in ALK-mutated cells expressing wild-type MYCN. Here, an inhibitor with potency against both mTOR and PI3K was more effective in promoting cytotoxicity when combined with crizotinib. Our findings should enable a more precise selection of molecularly targeted agents for patients with ALK-mutated tumors.