Driver Kinases Research Articles

The molecular basis of the dichotomous functionality of MAP4K4 in proliferation and cell motility control in cancer.

The finely tuned integration of intra- and extracellular cues by components of the mitogen-activated protein kinase (MAPK) signaling pathways controls the mutually exclusive phenotypic manifestations of uncontrolled growth and tumor cell dissemination. The Ser/Thr kinase MAP4K4 is an upstream integrator of extracellular cues involved in both proliferation and cell motility control. Initially identified as an activator of the c-Jun N-terminal kinase (JNK), the discovery of diverse functions and additional effectors of MAP4K4 beyond JNK signaling has considerably broadened our understanding of this complex kinase. The implication of MAP4K4 in the regulation of cytoskeleton dynamics and cell motility provided essential insights into its role as a pro-metastatic kinase in cancer. However, the more recently revealed role of MAP4K4 as an activator of the Hippo tumor suppressor pathway has complicated the understanding of MAP4K4 as an oncogenic driver kinase. To develop a better understanding of the diverse functions of MAP4K4 and their potential significance in oncogenesis and tumor progression, we have collected and assessed the current evidence of MAP4K4 implication in molecular mechanisms that control proliferation and promote cell motility. A better understanding of these mechanisms is particularly relevant in the brain, where MAP4K4 is highly expressed and under pathological conditions either drives neuronal cell death in neurodegenerative diseases or cell dissemination in malignant tumors. We review established effectors and present novel interactors of MAP4K4, which offer mechanistic insights into MAP4K4 function and may inspire novel intervention strategies. We discuss possible implications of novel interactors in tumor growth and dissemination and evaluate potential therapeutic strategies to selectively repress pro-oncogenic functions of MAP4K4.

Early Reduction of Glucose Consumption Is a Biomarker of Kinase Inhibitor Efficacy Which Can Be Reversed with GLUT1 Overexpression in Lung Cancer Cells.

Small molecule inhibitors that target oncogenic driver kinases are an important class of therapies for non-small cell lung cancer (NSCLC) and other malignancies. However, these therapies are not without their challenges. Each inhibitor works on only a subset of patients, the pharmacokinetics of these inhibitors is variable, and these inhibitors are associated with significant side effects. Many of these inhibitors lack non-invasive biomarkers to confirm pharmacodynamic efficacy, and our understanding of how these inhibitors block cancer cell growth remains incomplete. Limited clinical studies suggest that early (< 2weeks after start of therapy) changes in tumor glucose consumption, measured by [18F]FDG PET imaging, can predict therapeutic efficacy, but the scope of this strategy and functional relevance of this inhibition of glucose consumption remains understudied. Here we demonstrate that early inhibition of glucose consumption as can be measured clinically with [18F]FDG PET is a consistent phenotype of efficacious targeted kinase inhibitors and is necessary for the subsequent inhibition of growth across models of NSCLC. We tested nine NSCLC cell lines (A549, H1129, H1734, H1993, H2228, H3122, H460, HCC827, and PC9 cells) and ten targeted therapies (afatinib, buparlisib, ceritinib, cabozantinib, crizotinib, dovitinib, erlotinib, ponatinib, trametinib, and vemurafenib) across concentrations ranging from 1.6nM to 5µM to evaluate whether these inhibitors block glucose consumption at 24-h post-drug treatment and cell growth at 72-h post-drug treatment. We overexpressed the facilitative glucose transporter SLC2A1 (GLUT1) to test the functional connection between blocked glucose consumption and cell growth after treatment with a kinase inhibitor. A subset of these inhibitors and cell lines were studied in vivo. Across the nine NSCLC cell lines, ten targeted therapies, and a range of inhibitor concentrations, whether a kinase inhibitor blocked glucose consumption at 24-h post-drug treatment strongly correlated with whether that inhibitor blocked cell growth at 72-h post-drug treatment in cell culture. These results were confirmed in vivo with [18F]FDG PET imaging. GLUT1 overexpression blocked the kinase inhibitors from limiting glucose consumption and cell growth. Our results demonstrate that the early inhibition of lung cancer glucose consumption in response to a kinase inhibitor is a strong biomarker of and is often required for the subsequent inhibition of cell growth. Early inhibition of glucose consumption may provide complementary information to other biomarkers in determining whether a drug will effectively limit tumor growth.

Abstract 6207: Ba/F3 kinase mutant cell panel, a powerful cell model for the discovery and development of inhibitors for kinase specific mutation

Abstract Although over 70 protein kinase inhibitors have been approved by FDA since 2001, the protein kinase is still the hot target class of molecularly targeted cancer therapy because of the discovery of new driver kinase fusion mutation and a large number of secondary kinase mutations after the inhibitor treatment in cancer patients with the sequencing technology advancement, the need for new generation specific small molecule kinase inhibitors is still unmet, and it urge the industry to development more accurate, efficient kinase cellular assay models.Ba/F3 line is a popular cell model because many protein kinases and their mutants can transform the Ba/F3 cells to be growth independent of exogenous IL-3 supplement, while their inhibitors can antagonize these effects. Our group have generated a large of cell line panel with over 300 different well validated (sequencing, Western Blot and inhibitor test) Ba/F3 kinase cell lines for kinase inhibitor screening and development, covering several hot kinases, such as EGFR, EML-ALK, FLT3, NTRK, ROS1, HER, BCR-ABL, RET, FGFR1, FGFR2, FGFR3, FGFR4, KRAS, cMET, et al. Except used in in vitro high-throughput compound screening, most of the transformed Ba/F3 lines can grow well in immune-deficient mice and were also used to evaluate the efficacy of drug candidate and its downstream signaling in vivo. Our data indicate that our Ba/F3 cell line panel is a powerful cell model for new kinase inhibitor discovery and development. Citation Format: Yu Wang, Yao Peng, Yao Tang, Lin Zhou, Jinying Ning, Feng Hao. Ba/F3 kinase mutant cell panel, a powerful cell model for the discovery and development of inhibitors for kinase specific mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6207.

Integrated Phosphoproteomic and N-Glycoproteomic Landscape of Cutaneous T-Cell Lymphoma Reveals Distinctive Pathogenetic Insights and Therapeutic Vulnerabilities

Background: A critical goal of precision oncology is the identification of deregulated signaling pathways that serve as disease-specific biomarkers and vulnerabilities that may be exploited as therapeutic targets. Cutaneous T cell lymphomas (CTCLs) are heterogeneous neoplasms with variable clinical outcomes. Whereas the genomic landscapes of CTCLs are being characterized, a large scale analysis of their proteomic signatures and post-translational modifications (PTM) has not been performed. In this study, we sought to gain insights into the pathogenesis of CTCLs using high mass accuracy tandem mass spectrometry (HMA-MS/MS)-based phosphoproteomics and glycoproteomics.Methods: CTCL-derived cell lines representative of Sézary syndrome (SS, HUT78), mycosis fungoides (MF, HH), and ALK-negative anaplastic large cell lymphoma (ALCL) with PCM1-JAK2 or NPM1-TYK2 translocations (Mac1, Mac2a, and Myla) were analyzed in technical and biologic triplicates to perform unbiased proteome characterization. Phosphoproteins were enriched using metal oxide affinity chromatography and immunoprecipitation using 3 anti-phosphotyrosine antibodies and N-glycoproteins were enriched using solid phase extraction and hydrazide resin and an Orbitrap Fusion or LTQ OrbitrapXL in-line with a Paradigm MS2 HPLC. Precise site-mapping of the PTMs was assigned and quantitated using Maxquant. Hierarchical clustering was performed using Perseus. Whole exome sequencing (WES) was performed using an Illumina HiSeq 2500. Functional studies using tyrosine kinase inhibitors were performed to assess selective vulnerabilities.Results: Phosphoproteomic analysis identified 1899 tyrosine (Y) phosphoproteins and 6029 serine/threonine (S/T) phosphoproteins. Unsupervised hierarchical clustering of the Y-phosphoproteome data accurately grouped the CTCLs to those with and without underlying tyrosine kinase translocations. Unique driver kinase phosphosignaling signatures were identified in MYLA (NPM1-TYK2) and MAC1/2 (PCM1-JAK2) representing the ALCL subgroup, as well as close similarity between the signatures of MF and SS-derived cells (HH and HUT78). Notably, phosphoproteomic analysis of HUT78 (SS) yielded high spectral counts of Y-phosphorylated residues at IL2RG p.Y325, JAK3 p.Y980/Y981, and STAT5 p.Y699 indicative of constitutive activation of the JAK/STAT5 pathway corroborated by WES showing activating mutations in JAK3 p.A573V and JAK1 p.Y654F.Beyond driver kinases, oncogenic kinase-specific substrates with distinct site-specific phosphorylated residues were identified for each disease class. Phosphoproteins selectively expressed in MF/SS, (46 p-S/T and 25 p-Y proteins) included members of the killer-cell immunoglobulin-like receptor (KIR) family implicated in immune modulation through immune tyrosine-based inhibitor or TYRO protein tyrosine kinase binding domains for signal transduction. Additionally, proteins not previously associated with MF/SS including NKG2-D type integral membrane protein family members were discriminative of this subgroup. Phosphoprotein members of lysine demethylases (KDM family) and Aryl Hydrocarbon Receptor families were distinctively identified in MF/SS cells, while components of the T-cell receptor signaling pathways were common in all groups of CTCLs. By comparison, N-glycoproteomic analysis identified 811 unique N-glycoproteins. A total of 51 N-glycoproteins were found solely in HUT78 of which 46 were novel in their specificity and included 13 with a role in cell-adhesion while N-glycoproteins unique to HH revealed 12 novel candidate biomarkers (e.g. MERTK, CD97 and SLC44A2). Integrative phosphoproteomic and N-glycoproteomic analysis strikingly revealed convergent pathways implicating a prominent role for the KIR family proteins in SS. Orthogonal immunophenotyping by flow cytometry has shown high sensitivity (96%) detection of KIR3DL2 in primary human SS cases. Significantly, anti-KIR3DL2 inhibition using a humanized antibody revealed activity against SS cells. By comparison, TYK2-driven CTCL (MYLA) was sensitive to inhibitors of TYK2 and JAK.Conclusions: Integrated phosphoproteomic and N-glycoproteomic analysis identifies known and novel candidate biomarkers, unique PTM signatures, and therapeutic vulnerabilities implicating disease-specific pathogenetic mechanisms in CTCL. DisclosuresNo relevant conflicts of interest to declare.

Progress in Treating Advanced Thyroid Cancers in the Era of Targeted Therapy.

Background: Thyroid cancer is a common malignancy whose detection has increased significantly in past decades. Most of the increased incidence is due to detection of early well-differentiated thyroid cancer, but the incidence of more advanced thyroid cancers has increased as well. Recent methodological advancements have allowed for a deep understanding of the molecular underpinnings of the various types of thyroid cancer. Summary: Thyroid cancers harbor a high frequency of potential druggable molecular alterations, including the highest frequency of oncogenic driver kinase fusions seen across all solid tumors. Analyses of poorly differentiated and anaplastic thyroid carcinoma confirmed that these tumors develop from more well-differentiated follicular-derived thyroid cancers through acquired additional mutations. The recognition of driver genomic alterations in thyroid cancers not only predicts tumor phenotype but also now can inform treatment approaches. Conclusions: Major progress in understanding the oncogenic molecular underpinnings across the array of thyroid cancers has led to considerable gains in gene-specific systemic therapies for many cancers. This article focuses on the molecular characteristics of aggressive follicular-derived thyroid cancers and medullary thyroid cancer and highlights advancements in treating thyroid cancer in the era of targeted therapy.

New Therapeutic Opportunities for the Treatment of Squamous Cell Carcinomas: A Focus on Novel Driver Kinases.

Squamous cell carcinomas of the lung, head and neck, esophagus, and cervix account for more than two million cases of cancer per year worldwide with very few targetable therapies available and minimal clinical improvement in the past three decades. Although these carcinomas are differentiated anatomically, their genetic landscape shares numerous common genetic alterations. Amplification of the third chromosome’s distal portion (3q) is a distinguishing genetic alteration in most of these carcinomas and leads to copy-number gain and amplification of numerous oncogenic proteins. This area of the chromosome harbors known oncogenes involved in squamous cell fate decisions and differentiation, including TP63, SOX2, ECT2, and PIK3CA. Furthermore, novel targetable oncogenic kinases within this amplicon include PRKCI, PAK2, MAP3K13, and TNIK. TCGA analysis of these genes identified amplification in more than 20% of clinical squamous cell carcinoma samples, correlating with a significant decrease in overall patient survival. Alteration of these genes frequently co-occurs and is dependent on 3q-chromosome amplification. The dependency of cancer cells on these amplified kinases provides a route toward personalized medicine in squamous cell carcinoma patients through development of small-molecules targeting these kinases.

Mining potentially actionable kinase gene fusions in cancer cell lines with the KuNG FU database

Inhibition of kinase gene fusions (KGFs) has proven successful in cancer treatment and continues to represent an attractive research area, due to kinase druggability and clinical validation. Indeed, literature and public databases report a remarkable number of KGFs as potential drug targets, often identified by in vitro characterization of tumor cell line models and confirmed also in clinical samples. However, KGF molecular and experimental information can sometimes be sparse and partially overlapping, suggesting the need for a specific annotation database of KGFs, conveniently condensing all the molecular details that can support targeted drug development pipelines and diagnostic approaches. Here, we describe KuNG FU (KiNase Gene FUsion), a manually curated database collecting detailed annotations on KGFs that were identified and experimentally validated in human cancer cell lines from multiple sources, exclusively focusing on in-frame KGF events retaining an intact kinase domain, representing potentially active driver kinase targets. To our knowledge, KuNG FU represents to date the largest freely accessible homogeneous and curated database of kinase gene fusions in cell line models.

A Phase 1 a/b Dose Escalation Study of the Mutation Agnostic BTK/FLT3 Inhibitor CG-806 in Patients with Relapsed or Refractory CLL/SLL or Non-Hodgkin's Lymphomas

INTRODUCTION: CG-806 is a potent, non-covalent oral inhibitor of Bruton's tyrosine kinase (BTK), FMS-like tyrosine kinase 3 (FLT3) and certain other validated driver kinases of malignancies, while sparing kinases typically associated with clinical toxicity. In cell lines and primary samples from chronic lymphocytic leukemia (CLL) patients, CG-806 suppressed BCR signaling (LYN, SYK, BTK, AKT, ERK) and pathways operative in AML (FLT3, AKT, CSF1R, MAPK, PDGFRα, others), killed malignant B-cell cells insensitive to ibrutinib or venetoclax at low nM concentrations, and showed enhanced activity in combination with venetoclax. CG-806 is currently being evaluated in a Phase 1 a/b trial in patients with CLL and select relapsed or refractory B-cell malignancies (NCT03893682). A parallel Phase 1 a/b clinical study of CG-806 in patients with relapsed or refractory (R/R) FLT3-mutant or FLT3-wildtype AML is in progress (NCT04477291). AIMS: The primary objectives of the clinical study NCT03893682 are to assess the safety and tolerability of CG-806 and to determine the recommended Phase 2 dose for future clinical trials in patients with advanced CLL/small lymphocytic lymphoma (SLL) or non-Hodgkin lymphoma (NHL). Key secondary objectives include elucidation of the pharmacokinetic (PK) and pharmacodynamic (PD) characteristics, preliminary evidence of antitumor activity, and selection of a starting dose for a trial in patients with R/R AML. METHODS: Eligible patients are those with CLL/SLL or NHL for which either the standard treatment has failed, is no longer effective, or can no longer be administered safely. Treatment emergent adverse events (TEAEs) and tumor responses are evaluated per disease specific guidelines. CG-806 is administered as capsules PO BID in 28-day cycles. RESULTS: As of July 24, 2020, a total of 11 patients (age 66.2 ± 7.4 years, male 54.5%, female 45.5%, 5 CLL/SLL, 3 follicular lymphoma, 1 Richter transformation lymphoma, 1 Waldenstrom's macroglobulinemia, 1 mantle cell lymphoma) with a median of 7 prior systemic regimens have been treated with CG-806 at doses of 150 mg (n=1), 300 mg (n=1), 450 mg (n=5), and 600 mg (n=4). No dose limiting toxicities or related serious adverse event have been reported. The most common drug related or possible related TEAEs included diarrhea, nausea, neutropenia in 3 (27.3%) each; fatigue, lymphocytosis and vomiting in 2 (18.2%) patients each. Drug related or possible related grade 3 or greater TEAEs included diarrhea (n=1, 9.1%) and neutropenia (n=2, 18.2%), but no fevers were reported - those two neutropenia events occurred in a patient with Richter transformation from CLL who had failed 12 prior regimens including ibrutinib, venetoclax, binutuzumab, CAR-NK and CAR-T and was found to have a prior therapy-related myeloid neoplasm after enrollment. Among three classic CLL patients placed on study, all experienced on-target lymphocytosis upon initiation of CG-806 therapy, as well as inhibition of phospho-BTK measured in the whole blood by ELISA and measured by a plasma inhibitor activity (PIA) assay with reporter cells. Steady state plasma levels of CG-806 in patients treated with CG-806 at 300 mg or higher doses dramatically reduced phosphorylated BTK-Y551, BTK-Y223, SYK-Y525/526, ERK-T202/Y204 and FLT3-Y591 in EOL-1 reporter cells, as determined by PIA assay, documenting multi-target engagement. Preliminary PK analyses showed the steady-state (Cmin) plasma levels of CG-806 on cycle 2 day 1 in patients treated with ≥300 mg BID were generally in the 1µM range and were sustained over multiple cycles. CONCLUSIONS: CG-806 demonstrated a favorable safety profile in patients treated to date with 150, 300, 450 or 600 mg BID over multiple cycles. Oral absorption in patients produced plasma concentrations known to be effective in murine leukemia models. Pharmacodynamic studies using patient whole blood and plasma documented inhibition of SYK, BTK and ERK in the BCR signaling pathway. Further, the trial allowed selection of a potentially therapeutically active starting dose for AML patients (450mg). Enrollment of patients with R/R CLL/SLL and NHL at dose level 5 (750mg) is ongoing and updated clinical data will be presented at the meeting. Disclosures Bejar: Aptose Biosciences, Inc: Current Employment, Current equity holder in publicly-traded company. Zhang:Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company. Rastgoo:Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company. Benbatoul:Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company. Jin:Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company. Thayer:Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company. Sheng:Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company. Chow:Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company. Montalvo-Lugo:Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company. Marango:Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company. Howell:Aptose Biosciences, Inc.: Current equity holder in publicly-traded company. Rice:Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company.

Abstract 5636: Restoration of tumor immunosurveillance in tasmanian devil facial tumor disease

Abstract Transmissible cancer is a rare, but mechanistically not understood form of cancer that can directly spread between individuals. At least six species in the animal kingdom harbor clonal cancers that spread horizontally. These diseases include the fatal devil facial tumor disease (DFTD) in Tasmanian devils, a sexually transmitted sarcoma in dogs, and leukemia-like cancers in mollusks. DFTD is an allogeneic graft of Schwann cell origin, which is transmitted by direct transfer of cancer cells from one individual to another as a result of biting behavior. Two strains of DFTD, DFT1 and DFT2, emerged independently in the population of Tasmanian devils. It is essential to gain mechanistic understanding of horizontal cancers to understand how cancer cells escape host immune cells. We designed an integrative systems biology approach using primary biopsies, established Tasmanian devil tumor and control cell lines and mouse xenograft model. Specifically, we combined pharmacological drug screening, transcriptomics, epigenetics, proteomics and comparative pathology to unveil aberrant signaling pathways in DFTD. We found that DFT1 tumor cells express high levels of phosphorylated ERBB2 and ERBB3 tyrosine kinase receptor and they are exquisitely vulnerable to ERBB kinase inhibitors. Blockade of the hyperactive ERBB-STAT3 axis with ERBB inhibitor sapitinib or with the STAT3 inhibitor DR-1-55 led to recovery of MHC-I expression, suggesting that immune escape could be antagonized by blockade of hyperactive ERBB-STAT3 action. In contrary to DFT1, progression of DFT2 is known to be associated with PDGF receptor amplification. This proposed an attractive pharmacological approach of targeted inhibition of PDGFR by broad kinase inhibitor imatinib. We validated in xenograft mouse model that targeted inhibition of PDGFR results in the efficient reduction of tumor growth as well as restoration of MHC-I gene expression. In summary, we identified a critical role for hyperactive tyrosine kinase-STAT signaling whose pharmacological inhibition resulted in arrested growth and possible restoration of immune cell recognition. We validated that DFT2 displays a distinct oncogene mechanism than DFT1, but pharmacologic inhibition of hyperactive driver kinase pathway leads to similar restoration of MHC-I expression. This link between the hyperactive pathway and MHC-I-mediated tumor immunosurveillance in both DFTD strains provides mechanistic insights into horizontal transmissibility and suggests a strategy to save Tasmanian devils from extinction. Citation Format: Anna Orlova, Lindsay Kosack, Alexandra M. Popa, Csilla Viczenczova, Alexander Lercher, Elvin D. de Araujo, Patrick Gunning, Richard Moriggl, Andreas Bergthaler. Restoration of tumor immunosurveillance in tasmanian devil facial tumor disease [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5636.

Phosphotyrosine-based Phosphoproteomics for Target Identification and Drug Response Prediction in AML Cell Lines

Acute myeloid leukemia (AML) is a clonal disorder arising from hematopoietic myeloid progenitors. Aberrantly activated tyrosine kinases (TK) are involved in leukemogenesis and are associated with poor treatment outcome. Kinase inhibitor (KI) treatment has shown promise in improving patient outcome in AML. However, inhibitor selection for patients is suboptimal.In a preclinical effort to address KI selection, we analyzed a panel of 16 AML cell lines using phosphotyrosine (pY) enrichment-based, label-free phosphoproteomics. The Integrative Inferred Kinase Activity (INKA) algorithm was used to identify hyperphosphorylated, active kinases as candidates for KI treatment, and efficacy of selected KIs was tested.Heterogeneous signaling was observed with between 241 and 2764 phosphopeptides detected per cell line. Of 4853 identified phosphopeptides with 4229 phosphosites, 4459 phosphopeptides (4430 pY) were linked to 3605 class I sites (3525 pY). INKA analysis in single cell lines successfully pinpointed driver kinases (PDGFRA, JAK2, KIT and FLT3) corresponding with activating mutations present in these cell lines. Furthermore, potential receptor tyrosine kinase (RTK) drivers, undetected by standard molecular analyses, were identified in four cell lines (FGFR1 in KG-1 and KG-1a, PDGFRA in Kasumi-3, and FLT3 in MM6). These cell lines proved highly sensitive to specific KIs. Six AML cell lines without a clear RTK driver showed evidence of MAPK1/3 activation, indicative of the presence of activating upstream RAS mutations. Importantly, FLT3 phosphorylation was demonstrated in two clinical AML samples with a FLT3 internal tandem duplication (ITD) mutation.Our data show the potential of pY-phosphoproteomics and INKA analysis to provide insight in AML TK signaling and identify hyperactive kinases as potential targets for treatment in AML cell lines. These results warrant future investigation of clinical samples to further our understanding of TK phosphorylation in relation to clinical response in the individual patient.

Non-small cell lung cancer-small cell lung cancer transformation as mechanism of resistance to tyrosine kinase inhibitors in lung cancer

Mutated or rearranged driver kinases in non-small cell lung cancer (NSCLC) cells are clinically amenable to treatment with tyrosine kinase inhibitors (TKIs) resulting in prolonged survival and significant benefit compared to cytotoxic chemotherapy. The most frequent genomic alterations are observed for epidermal growth factor receptor and anaplastic lymphoma kinase, which can be blocked by a range of specific TKIs in sequence. In clinics, resistance to TKIs emerges after approximately one year and comprises secondary mutations of the kinases (on-target) or alternative pathways circumventing the original kinase (off-target) alterations. A special feature of NSCLC is the occurrence of histological transformation to small cell lung cancer (SCLC) in up to 14% of cases, which, in general, is accompanied by resistance to the original TKIs. SCLC transformed tumors may be treated with the classical platinum/etoposide regimen but thus far there are no definitive guidelines. Four transformed pleural SCLC lines in our lab indicate the presence of a gradual NSCLC-SCLC shift with overlapping drug sensitivities. In conclusion, the treatment of NSCLC-SCLC transformed cancer cells would need a better chemosensitivity assessment using functional genomics to guide further therapy.

2039P - Driving solo? Investigation into collaborating mutations in SDH-deficient neoplasia

BackgroundSuccinate dehydrogenase (SDH) is a highly conserved component of energy production and metabolism. Genetic or epigenetic inactivation of SDH (dSDH) creates a state of intracellular pseudohypoxia that is uniquely oncogenic for subtypes of GIST, renal cell carcinoma (RCC), paraganglioma/pheochromocytoma (PGL/Pheo), and pituitary adenoma/carcinoma (PAC). dSHD stems from homozygous loss of one of four subunit encoding genes SDHA, SDHB, SDHC, or SDHD (aka, SDHx). dSDH is not targetable, and preclinical models are poorly viable. In the current study we analyze comprehensive genomic profiles (CGP) from a dSDH pan-tumor cohort for potential therapy-enabling non-SDHx genomic alterations and biomarkers (GA). Methods231,706 clinical grade CGP were searched for dSDH GIST, RCC, PGL/Pheo, and PAC. GA were determined on 1.1Mb genome sequence encompassing up to 324 cancer genes. dSDH was further established by histopathology review and analysis of SDHx homozygous loss. Germline and zygosity status of SDHx variants were performed by SGZ algorithm. Non-SDHx GA were assessed for therapeutic actionability. Results82 SDH-deficient neoplasms were analyzed (Table). 0 of 38 GIST had highly actionable GA. In one case a subclonal KIT variant was identified in a recurrence of an SDHA-mutant GIST treated with Imatinib for several years. No clonal canonical driver kinase mutations were identified in KIT, PDGFRA, NF1, BRAF, FGFR1 or NTRK1-3. 0 of 36 dSDH PGL/Pheo and 0 of 1 PAC had potential therapeutic options. 2 of 7 dSDH RCC reported potential treatment options for oncogenic variants in EGFR and CDK4. No dSDH tumors had high TMB, microsatellite instability, or LOH score indicative of homologous recombination defect (HRD). Overall, 2 of 82 tumors (2.4%) harbored highly actionable GA.Table2039PTable82 SDH-deficient tumorsGIST (n=38)PGL/Pheo (n=36)RCC (n=7)PAC (n=1)Age (mean)33404944Sex ratio (M/F)16/2124/124/30/1SDHA111231SDHB72130SDHC4110SDHD2200SDHx-WT14000%SDHx germline696410050GA/tumor (pathogenic non-SDHx)0.580.892.290MSI0000TMB (mut/Mb, mean)1.862.430.9HRD (LOHscore >16%)0000 ConclusionsThis study identifies SDH deficiency as a lone driver of malignancy and without associated actionable GA in>97% of cases, and underscores the need for novel therapeutic approaches targeting SDH deficiency itself. Legal entity responsible for the studyFoundation Medicine. FundingFoundation Medicine. DisclosureJ.K. Killian: Full / Part-time employment: Foundation Medicine. D.C. Pavlick: Full / Part-time employment: Foundation Medicine. E.S. Sokol: Full / Part-time employment: Foundation Medicine. M. Montesion: Full / Part-time employment: Foundation Medicine. D.X. Jin: Full / Part-time employment: Foundation Medicine. B. Kaplan: Full / Part-time employment: Foundation Medicine. D. Lin: Full / Part-time employment: Foundation Medicine. J. Vergilio: Full / Part-time employment: Foundation Medicine. J.A. Elvin: Full / Part-time employment: Foundation Medicine. N. Ngo: Full / Part-time employment: Foundation Medicine. E. Severson: Full / Part-time employment: Foundation Medicine. S. Ramkissoon: Full / Part-time employment: Foundation Medicine. D. Duncan: Full / Part-time employment: Foundation Medicine. C. Edgerly: Full / Part-time employment: Foundation Medicine. A. Hemmerich: Full / Part-time employment: Foundation Medicine. G.M. Frampton: Full / Part-time employment: Foundation Medicine. V.A. Miller: Full / Part-time employment: Foundation Medicine. S.M. Ali: Full / Part-time employment: Foundation Medicine. J.S. Ross: Full / Part-time employment: Foundation Medicine. All other authors have declared no conflicts of interest.

First-time in-human study of VMD-928, an allosteric and irreversible TrkA selective inhibitor, in patients with solid tumors or lymphoma.

TPS3146 Background: Tropomysin receptor kinase A (TrkA) is a protein encoded by the NTRK1 gene. NTRK fusions involving the kinase domain are oncogenic for multiple tumor types and larotrectinib was recently approved for advanced solid tumors harboring NTRK gene fusions. Larotrectinib, an ATP-competitive, reversible pan-TrkA/B/C inhibitor, has shown impressive response rates in patients harboring these fusions; however, resistance can develop due to acquired ATP-site mutations. This has been previously identified in other oncogenic driver kinases such as ALK and EGFR treated with ATP-competitive kinase inhibitors. A newly approved allosteric ALK/EGFR inhibitor brigatinib was able to clinically overcome acquired resistance of many ATP-competitive ALK/EGFR inhibitors (1). Also, irreversible EGFR inhibitors such as afatinib (ATP-competitive) were active against tumors resistant to first-generation inhibitors (2), although their efficacy can be compromised by acquired ATP-site mutations (3). VMD-928 is the first oral small-molecule TrkA (NTRK1) selective inhibitor with dual allosteric and irreversible mechanisms of action. It inhibits TrkA non-competitively at an allosteric (non-ATP) site and has no resistance in vitro to acquired ATP-site mutations such as G667C. VMD-928 in vitro has little or no activity against 348 other kinases including TrkB (NTRK2) and TrkC (NTRK3). We are conducting the first time in human phase 1 trial of oral VMD-928, a novel allosteric and irreversible TrkA selective inhibitor. Methods: This is an open label, Phase 1 study investigating the safety, pharmacokinetics (PK) and pharmacodynamics (PD) of oral VMD-928 in adults with advanced solid tumors or lymphoma (NCT03556228). In part 1 of the study, an accelerated titration scheme will be utilized to determine the recommended phase 2 dose and evaluate PK / PD of VMD-928. In part 2, expansion cohorts including patients with thymic, pancreatic, triple-negative breast carcinoma, or solid tumors with TrkA alterations will be accrued to further evaluate safety and efficacy. Part 3 of the study will characterize the biologically active dose. The study is open and accruing patients at City of Hope. Clinical trial information: NCT03556228.

Abstract P2-06-25: A phenotypic screening and machine learning platform efficiently identifies triple negative breast cancer-selective and readily druggable targets

Abstract Identifying effective oncogenic targets is challenged by the complexity of genetic alterations in cancer and their poorly understood relation to cell function and survival. This, combined with the fact that the majority of the proteome is predicted to be undruggable, may explain why genomic approaches have had only moderate success in identifying and translating oncogenic drug targets. There is a need for methods that rapidly and accurately identify “pharmacologically effective” targets without the requirement for priori knowledge of complex signaling networks. We developed an approach that uses machine learning to relate results from unbiased phenotypic screening of kinase inhibitors to their biochemical activity data. This process, which we call idTRAX (Identification of Drug TaRgets and Anti-targets by Cellular and Molecular Cross-referencing), identifies targets that are pharmacologically responsive and readily druggable. Additionally, the identified targets are not typically overcome by the robustness of signaling networks, because only targets that effectively induce a phenotype upon pharmacological engagement are selected. We applied this methodology to triple negative breast cancer, which still lacks targeted therapy. We screened 19 breast cancer cell lines with ˜500 small-molecule kinase inhibitors with annotated kinase activity data and identified cell-line specific kinase targets, i.e. kinases whose inhibition causes cell line-specific cytotoxicity or cytostaticity. We were able to identify various unique cell line selective kinase addictions along with well-known driver kinase addictions, for example, dependence on FGFR2 in the MFM-223 cell line and AKT kinases in MFM-223, CAL-148, and others. We also found that triple negative breast cancer cell lines exhibit heterogeneous target patterns, indicating the need of personalized medicine approach to tackle them. We further compared candidate targets identified by idTRAX with those reported from RNAi- and CRISPR-based screens. Strikingly, the correlation between targets identified by the pharmacological approach and those identified by knockdown/knockout-based screens was low [average rank correlation (ρ) = 0.0273- 0.1074], suggesting that gene-silencing approaches may not be the most efficient at identifying targets for small-molecule drug discovery or repurposing. Our approach provides a platform for rapidly identifying sample-specific drug targets and potentially guiding personalised therapy regimens. Citation Format: Gautam P, Jaiswal A, Aittokallio T, Hassan A-A, Wennerberg K. A phenotypic screening and machine learning platform efficiently identifies triple negative breast cancer-selective and readily druggable targets [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-25.

Preclinical Evaluation of Nintedanib, a Triple Angiokinase Inhibitor, in Soft-tissue Sarcoma: Potential Therapeutic Implication for Synovial Sarcoma.

Sarcomas are rare cancers that make up about 1% of all cancers in adults; however, they occur more commonly among children and young adolescents. Sarcomas are genetically complex and are often difficult to treat given the lack of clinical efficacy of any of the currently available therapies. Receptor tyrosine kinases (RTK) such as c-Kit, c-Met, PDGFR, IGF-1R, as well as FGFR have all been reported to be involved in driving tumor development and progression in adult and pediatric soft-tissue sarcoma. These driver kinases often act as critical determinants of tumor cell proliferation and targeting these signal transduction pathways remains an attractive therapeutic approach. Nintedanib, a potent triple angiokinase inhibitor, targets PDGFR, VEGFR, and FGFR pathways critical for tumor angiogenesis and vasculature. In this study, we evaluated the preclinical efficacy of nintedanib in soft-tissue sarcoma cell lines. Nintedanib treatment resulted in significant antiproliferative effect in vitro in cell lines with high expression of RTK drug targets. Furthermore, treatment with nintedanib showed significant downregulation of downstream phosphorylated AKT and ERK1/2. Finally, treatment with nintedanib resulted in significant tumor growth suppression in mouse xenograft model of synovial sarcoma. Notably, both the in vitro and in vivo efficacy of nintedanib was superior to that of imatinib, another multikinase inhibitor, previously tested with minimal success in clinical trials in sarcoma. Overall, the data from this study provide a strong rationale to warrant further clinical exploration of this drug in patients with synovial sarcoma. Mol Cancer Ther; 17(11); 2329-40. ©2018 AACR.

ES04.03 Overcoming Resistance by Improving Treatment Compliance

ES04.03 Overcoming Resistance by Improving Treatment Compliance

Abstract 4010: Identification of signaling pathways involved in colorectal adenoma-to-carcinoma progression using phosphoproteomics

Abstract Background Colorectal cancer (CRC) develops in a multi-step-process from normal epithelium, through a pre-malignant lesion (adenoma), into a malignant lesion (adenocarcinoma). A minority of about 5% of adenomas will ultimately progress into cancer. To improve clinical practice it is important to identify the subgroup of colorectal adenomas that is at risk of progressing to cancer. By extensive genomic analysis we have shown that specific copy number alterations are associated with risk of progression. An example of such an alteration is the gain of chromosome arm 20q and we identified AURKA and TPX2 as major drivers of this amplicon. Yet, the activation status of these and other genes during colorectal carcinogenesis and the downstream signaling pathways affected with gene (in)activation are not fully known. In order to better understand the biology of adenoma to carcinoma transition, we performed a comprehensive analysis of phosphoproteomes at different stages of colorectal carcinogenesis. Methods Phosphotyrosine containing peptides were immunoprecipitated from 5 mg of colorectal adenoma (n=81) and colorectal carcinoma tissues (n=50) using agarose bead-coupled phosphotyrosine antibody P-Tyr-1000. A Q Exactive HF mass spectrometer was used to perform NanoLC-MS/MS. Spectral counts of phosphoproteins and ion intensities of phosphopeptides were defined by MaxQuant for relative quantitation of protein phosphorylation. Results Phosphotyrosine-based phosphoproteomics of the 131 colorectal tissue samples yielded 6056 phosphopeptides, corresponding to 2745 unique phosphoproteins including 183 phosphokinases. Data analysis to identify regulated phosphorylation states and inference of kinase activity is ongoing. Conclusions and future plans This is the first large phosphoproteomics dataset of colorectal adenomas and carcinomas. Preliminary data analysis shows promising differences in kinase activities between adenomas and carcinomas. After further analysis, we will manipulate the activity of the driver kinases in adenoma-derived organoids and monitor tumor outgrowth in mice. Citation Format: Sanne Martens-de Kemp, Alex Henneman, Richard de Goeij-de Haas, Sander Piersma, Thang Pham, Gerrit Meijer, Beatriz Carvalho, Connie Jimenez. Identification of signaling pathways involved in colorectal adenoma-to-carcinoma progression using phosphoproteomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4010.

Phosphoproteomic Profiling Reveals ALK and MET as Novel Actionable Targets across Synovial Sarcoma Subtypes.

Despite intensive multimodal treatment of sarcomas, a heterogeneous group of malignant tumors arising from connective tissue, survival remains poor. Candidate-based targeted treatments have demonstrated limited clinical success, urging an unbiased and comprehensive analysis of oncogenic signaling networks to reveal therapeutic targets and personalized treatment strategies. Here we applied mass spectrometry-based phosphoproteomic profiling to the largest and most heterogeneous set of sarcoma cell lines characterized to date and identified novel tyrosine phosphorylation patterns, enhanced tyrosine kinases in specific subtypes, and potential driver kinases. ALK was identified as a novel driver in the Aska-SS synovial sarcoma (SS) cell line via expression of an ALK variant with a large extracellular domain deletion (ALKΔ2-17). Functional ALK dependency was confirmed in vitro and in vivo with selective inhibitors. Importantly, ALK immunopositivity was detected in 6 of 43 (14%) of SS patient specimens, one of which exhibited an ALK rearrangement. High PDGFRα phosphorylation also characterized SS cell lines, which was accompanied by enhanced MET activation in Yamato-SS cells. Although Yamato-SS cells were sensitive to crizotinib (ALK/MET-inhibitor) but not pazopanib (VEGFR/PDGFR-inhibitor) monotherapy in vitro, synergistic effects were observed upon drug combination. In vivo, both drugs were individually effective, with pazopanib efficacy likely attributable to reduced angiogenesis. MET or PDGFRα expression was detected in 58% and 84% of SS patients, respectively, with coexpression in 56%. Consequently, our integrated approach has led to the identification of ALK and MET as promising therapeutic targets in SS. Cancer Res; 77(16); 4279-92. ©2017 AACR.

Abstract 2378: A kinome analysis of the molecular pharmacodynamics of PT-112 in a human cancer cell line

Abstract Background: To better understand the mechanism of action of PT-112, we investigated the effects of PT-112 on kinase activity within HCT116 human colorectal cancer cells. PT-112 is a pyrophosphate-conjugated platinum (Pt) containing new chemical entity under clinical development in the US and Taiwan. Designed to reduce certain toxicities associated with Pt agents and drug resistance from DNA-repair pathway activity, PT-112 differs in significant ways from traditional Pt therapies. PT-112 results in potent anti-proliferation effects with lower Pt accumulation in whole cell and DNA extracts. Upregulation of p16 and p21 and, consistently, inhibition of the cell cycle at the G1/S phase transition is observed with PT-112 use, a finding not commonly associated with Pt agents. Further, sensitivity to PT-112 is not significantly affected by the functionality of DNA repair pathways. Finally, the induction of immunogenic cell death by PT-112 has been indicated via release of HMGB1, cell surface localization of calreticulin, and suppression of STAT3. Methods: A broad analysis of the phosphorylation pattern of kinase targets was achieved by utilizing the Tyrosine Kinase and Serine / Threonine Kinase PamChip® Arrays, each spotted with 144 peptides harboring kinase phosphorylation sites. Data were collected after 1, 6, and 24hr of exposure to 10 μM PT-112, a concentration achievable in human patient plasma at sub-toxic doses, and the degree of phosphorylation of these peptide targets was measured by the PamStation®. Results: A set of kinase targets was identified as differentially phosphorylated relative to untreated control samples. In total, 4/15/30 targets were more phosphorylated than controls at the 1/6/24hr time points, respectively, while 1/0/10 targets were less phosphorylated. Several of these targets connect to key cancer relevant pathways, including anti-angiogenesis (VGFR1), apoptosis (BAD), cell cycle regulation (CDC2, Rb, RBL2), cell growth (EGFR, MK07, RBL2), and cancer immunology (STAT4, CSF1R). Conclusions: A narrow set of initial effects on kinase activity from PT-112 exposure, which broaden over time, was observed. While we cannot discriminate direct from indirect effects, these data are consistent with PT-112 having mechanistic targets independent of nuclear DNA. Several observations were consistent with findings from previous experiments (e.g. evidence of impacts on apoptotic, cell cycle, and immunological pathways), while others were novel (e.g. impact on VGFR1, EGFR, MK07). In conclusion, PT-112 at pharmacologically achievable concentrations leads to a unique pattern of kinase disruption in vitro. Further work will be conducted to validate these findings, identify the driver kinases, and connect these observations into a coherent network of kinases and targets affected by PT-112 treatment, and to understand the clinical impact of the unique pharmacodynamic profile of PT-112. Citation Format: Diego Tosi, Esther Perez-Gracia, Philippe Pourquier, Tyler D. Ames, Richard A. Wing, José Jimeno, Céline Gongora. A kinome analysis of the molecular pharmacodynamics of PT-112 in a human cancer cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2378. doi:10.1158/1538-7445.AM2017-2378

Abstract 5837: Phosphoproteomic landscape of sarcoma: novel subgroups and actionable targets

Abstract Sarcomas comprise a heterogeneous group of malignant tumors of the connective tissue, and disproportionally affect the young. Despite intensive, multimodal treatments, survival is still limited and accompanied by many early and late side effects. In contrast to advances in carcinomas, specific targetable driver mutations have not or rarely been found in sarcoma and targeted therapies have met with disappointing clinical results, stressing the need for an unbiased and comprehensive analysis of oncogenic signaling networks to reveal therapeutic targets and personalized treatment strategies. We utilized a mass-spectrometry (MS)-based phosphoproteomics screening approach to profile the largest and most heterogeneous set of sarcoma cell lines (n=20) to date, including both adult (gastrointestinal stromal tumor (GIST) and angiosarcoma (AS)) and multiple pediatric/adolescent and young adult (AYA; synovial sarcoma (SS), Ewing sarcoma (ES) and rhabdomyosarcoma (RMS)) sarcomas, as well as soft-tissue sarcomas (STS) and bone sarcomas. We specifically included cell lines and SS and AS subtypes that were never phosphoprofiled before, to reveal completely novel molecular insights into sarcoma pathology. Validations were performed in vitro, in vivo, and on patient tumor material. Our unique tyrosine phosphorylation data revealed a novel subclassification of sarcoma, and showed that particularly the pediatric/AYA sarcoma models did not cluster within their histological subgroup. Particular tyrosine kinases exhibited enhanced phosphorylation in particular subtypes, and we identified driver kinases with outlier levels of activation. ALK was identified as a completely novel and unexpected driver in the Aska-SS SS cell line, which was explained by an underlying ALK translocation. Functional ALK dependency was confirmed with in vitro proliferation assays, showing high sensitivity for the ALK inhibitor TAE684 (IC50 26±3 nM) and the ALK/MET inhibitor crizotinib (IC50 46±5 nM). Patient specimens confirmed a clinical role for ALK in SS, with ALK immunopositivity in 14% (6/43) of patients and one 16y old patient harboring an ALK translocation, present in both her primary tumor as in 4 metastases. Other SS cell lines and sarcoma subtypes exhibited multiple activated tyrosine kinases, providing a rationale for combined targeting approaches. Indeed, high MET and PDGFRα phosphorylation in Yamato-SS SS cells predicted sensitivity to crizotinib and pazopanib (PDGFR) in vitro and in vivo. The clinical significance was again confirmed in a cohort of SS patients, in which MET or PDGFRα expression was detected in 58% and 84% of SS patients, respectively, with co-expression in 56%. Altogether, this is the first study to provide a deep insight into activated networks and molecular clustering patterns in sarcoma. Our integrated approach systematically identified and validated ALK and MET as novel and actionable drivers in SS. Citation Format: Emmy D. Fleuren, Myrella Vlenterie, Winette van der Graaf, Melissa Hillebrandt-Roeffen, Sabri Cebeci, Amy van de Ven, Uta Flucke, Jianmin Wu, Yvonne Versleijen-Jonkers, Roger Daly. Phosphoproteomic landscape of sarcoma: novel subgroups and actionable targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5837. doi:10.1158/1538-7445.AM2017-5837