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Prior to the early 2000s, patients with advanced gastrointestinal stromal tumors (GIST) had very poor prognoses owing to a lack of effective therapies. The development of tyrosine kinase inhibitors at the turn of the century significantly improved the overall survival for patients with GIST. The resounding success of imatinib in the first clinical trial of a tyrosine kinase inhibitor to treat GIST led to its approval for first-line therapy for advanced GIST; this study was open to all comers and not restricted to any GIST subtype(s). The trials that led to the approvals of second-, third-, and fourth-line therapy for advanced GIST were also open to all patients with advanced/metastatic GIST. Only in retrospect do we realize the role that the molecular subtypes played in the results observed in these studies. In this review, we discuss the studies that led to the US Food and Drug Administration approval of imatinib (first line), sunitinib (second line), regorafenib (third line), and ripretinib (fourth line) for advanced KIT-mutant GIST. In addition, we review how information about GIST molecular subtypes has been used to accelerate the approval of other targeted therapies for non-KIT mutant GIST, leading to the approval of five additional drugs indicated for the treatment of specific GIST molecular subtypes. We also discuss how our understanding of the molecular subtypes will play a role in the next generation of therapeutic approaches for treating advanced GIST.

Figure S1: HMC1.2 cell line has constitutively actived NFAT2, NFAT2 and NFAT4 species; Figure S2: RT-PCR of transcripts modulated by 24 hour treatment with CSA; Figure S3: CNPIs do not modulate KIT signaling and KIT inhibitors do not affect NFAT localization; Figure S4: Combination therapy does not enhance the effect of KIT inhibitors on KIT or downstream signaling; Table S1: Summary of KIT-mutant mast cell characteristics; Table S2: Summary of antibodies used in immunoblotting experiments; Table S3: qRT-PCR analysis of NFAT expression in KIT-mutant mast cell lines.

Gastrointestinal stromal tumors (GIST) harboring activating mutations of PDGFRA respond to imatinib, with the notable exception of the most common mutation, D842V. Avapritinib is a novel, potent KIT/PDGFRA inhibitor with substantial clinical activity in patients with the D842V genotype. To date, only a minority of PDGFRA-mutant patients treated with avapritinib have developed secondary resistance. Tumor and plasma biopsies in 6 of 7 patients with PDGFRA primary mutations who progressed on avapritinib or imatinib had secondary resistance mutations within PDGFRA exons 13, 14, and 15 that interfere with avapritinib binding. Secondary PDGFRA mutations causing V658A, N659K, Y676C, and G680R substitutions were found in 2 or more patients each, representing recurrent mechanisms of PDGFRA GIST drug resistance. Notably, most PDGFRA-mutant GISTs refractory to avapritinib remain dependent on the PDGFRA oncogenic signal. Inhibitors that target PDGFRA protein stability or inhibition of PDGFRA-dependent signaling pathways may overcome avapritinib resistance.Significance:Here, we provide the first description of avapritinib resistance mechanisms in PDGFRA-mutant GIST.This article is highlighted in the In This Issue feature, p. 1

Contains: Supplemental Figures 1-7; Supplemental Tables 1 -3; Supplemental Methods

Resistant KIT mutations have hindered the development of KIT kinase inhibitors for treatment of patients with systemic mastocytosis. The goal of this research was to characterize the synergistic effects of a novel combination therapy involving inhibition of KIT and calcineurin phosphatase, a nuclear factor of activated T cells (NFAT) regulator, using a panel of KIT-mutant mast cell lines. The effects of monotherapy or combination therapy on the cellular viability/survival of KIT-mutant mast cells were evaluated. In addition, NFAT-dependent transcriptional activity was monitored in a representative cell line to evaluate the mechanisms responsible for the efficacy of combination therapy. Finally, shRNA was used to stably knockdown calcineurin expression to confirm the role of calcineurin in the observed synergy. The combination of a KIT inhibitor and a calcineurin phosphatase inhibitor (CNPI) synergized to reduce cell viability and induce apoptosis in six distinct KIT-mutant mast cell lines. Both KIT inhibitors and CNPIs were found to decrease NFAT-dependent transcriptional activity. NFAT-specific inhibitors induced similar synergistic apoptosis induction as CNPIs when combined with a KIT inhibitor. Notably, NFAT was constitutively active in each KIT-mutant cell line tested. Knockdown of calcineurin subunit PPP3R1 sensitized cells to KIT inhibition and increased NFAT phosphorylation and cytoplasmic localization. Constitutive activation of NFAT appears to represent a novel and targetable characteristic of KIT-mutant mast cell disease. Our studies suggest that combining KIT inhibition with NFAT inhibition might represent a new treatment strategy for mast cell disease. Mol Cancer Ther; 13(12); 2840–51. ©2014 AACR.

Figure and table legends for supplemental figures 1-4 and supplemental tables 1-3.

Purpose: Patients who inherit a pathogenic loss-of-function genetic variant involving one of the four succinate dehydrogenase (SDH) subunit genes have up to an 86% chance of developing one or more cancers by the age of 50. If tumors are identified and removed early in these high-risk patients, they have a higher potential for cure. Unfortunately, many alterations identified in these genes are variants of unknown significance (VUS), confounding the identification of high-risk patients. If we could identify misclassified SDH VUS as benign or pathogenic SDH mutations, we could better select patients for cancer screening procedures and remove tumors at earlier stages.Experimental Design: In this study, we combine data from clinical observations, a functional yeast model, and a computational model to determine the pathogenicity of 22 SDHA VUS. We gathered SDHA VUS from two primary sources: The OHSU Knight Diagnostics Laboratory and the literature. We used a yeast model to identify the functional effect of a VUS on mitochondrial function with a variety of biochemical assays. The computational model was used to visualize variants' effect on protein structure.Results: We were able to draw conclusions on functional effects of variants using our three-prong approach to understanding VUS. We determined that 16 (73%) of the alterations are actually pathogenic, causing loss of SDH function, and six (27%) have no effect upon SDH function.Conclusions: We thus report the reclassification of the majority of the VUS tested as pathogenic, and highlight the need for more thorough functional assessment of inherited SDH variants. Clin Cancer Res; 23(21); 6733–43. ©2017 AACR.

TIFF file for supplemental figure 1

TIFF file for supplemental figure 1

Video for Biochemical, molecular, and clinical characterization of SDHA VUS from Biochemical, Molecular, and Clinical Characterization of Succinate Dehydrogenase Subunit A Variants of Unknown Significance

Supplemental tables 1 and 2, supplemental figure legends, supplemental video legend

Generative artificial intelligence (AI) has the potential to greatly increase the speed, quality and controllability of antibody design. Traditionalde novoantibody discovery requires time and resource intensive screening of large immune or synthetic libraries. These methods also offer little control over the output sequences, which can result in lead candidates with sub-optimal binding and poor developability attributes. Several groups have introduced models for generative antibody design with promisingin silicoevidence [1–10], however, no such method has demonstrated generative AI-basedde novoantibody design with experimental validation. Here we use generative deep learning models tode novodesign antibodies against three distinct targets, in azero-shotfashion, where all designs are the result of a single round of model generations with no follow-up optimization. In particular, we screen over 1 million antibody variants designed for binding to human epidermal growth factor receptor 2 (HER2) using our high-throughput wet lab capabilities. Our models successfully design all CDRs in the heavy chain of the antibody and compute likelihoods that are calibrated with binding. We achieve binding rates of 10.6% and 1.8% for heavy chain CDR3 (HCDR3) and HCDR123 designs respectively, which is four and eleven times higher than HCDR3s and HCDR123s randomly sampled from the Observed Antibody Space (OAS) [11]. We further characterize 421 AI-designed binders using surface plasmon resonance (SPR), finding three that bind tighter than the therapeutic antibody trastuzumab. The binders are highly diverse, have low sequence identity to known antibodies, and adopt variable structural conformations. Additionally, the binders score highly on our previously introducedNaturalnessmetric [12], indicating they are likely to possess desirable developability profiles and low immunogenicity. We open source1the HER2 binders and report the measured binding affinities. These results unlock a path to accelerated drug creation for novel therapeutic targets using generative AI and high-throughput experimentation.

When gastrointestinal stromal tumour (GIST), the most common form of sarcoma, was first recognized as a distinct pathological entity in the 1990s, patients with advanced-stage disease had a very poor prognosis owing to a lack of effective medical therapies. The discovery of KIT mutations as the first and most prevalent drivers of GIST and the subsequent development of the first KIT tyrosine kinase inhibitor (TKI), imatinib, revolutionized the treatment of patients with this disease. We can now identify the driver mutation in 99% of patients with GIST via molecular diagnostic testing, and therapies have been developed to treat many, but not all, molecular subtypes of the disease. At present, seven drugs are approved by the FDA for the treatment of advanced-stage GIST (imatinib, sunitinib, regorafenib, ripretinib, avapritinib, larotrectinib and entrectinib), all of which are TKIs. Although these agents can be very effective for treating certain GIST subtypes, challenges remain and new therapeutic approaches are needed. In this Review, we discuss the molecular subtypes of GIST and the evolution of current treatments, as well as their therapeutic limitations. We also highlight emerging therapeutic approaches that might overcome clinical challenges through novel strategies predicated on the biological features of the distinct GIST molecular subtypes.

Gastrointestinal stromal tumor (GIST) is the most common GI sarcoma and frequently harbor oncogenic mutations in KIT or platelet derived growth factor receptor alpha (PDGFRA) receptor tyrosine kinases. While type II tyrosine kinase inhibitors (TKIs) revolutionized the treatment of KIT-mutant GIST, they are thought to be ineffective for many PDGFRA-mutant GIST cases. Over 80% of PDGFRA mutations in GIST occur in exon 18 and patients with the most common mutation, D842V, are resistant to imatinib and other type II TKIs, leading to poor outcomes. However, beyond D842V, numerous other PDGFRA exon 18 mutations have been observed. From curated data of a large cohort of PDGFRA-mutant GISTs, we have identified nearly 50 unique PDGFRA exon 18 mutations, including both point mutations and complex indels. Although it is largely unknown how these mutations respond to imatinib, currently most, if not all, PDGFRA exon 18 mutations are assumed to be imatinib resistant. Using cell models, we tested some of the most commonly observed mutations in this cohort. Contrary to dogma, we observed that some exon 18 mutations tested were imatinib sensitive. We also observed that imatinib sensitivity seemed to be dependent on the properties of the residue in the 842-codon position, which encodes a key auto-inhibitory residue. We then hypothesized that imatinib sensitivity of every known or future reported PDGFRA exon 18 mutation might be predicted based on the 842 residue. To test the predictive potential of the 842 residue, we created mutagenic libraries that included every amino acid substitution at the 842 position. We expressed each mutation in Ba/F3 cells and assessed imatinib sensitivity via immunoblotting for phosphorylated-PDGFRA. Overall, 40% of tested mutations were sensitive to imatinib, further debunking the belief that all PDGFRA exon 18 mutations are imatinib resistant. In agreement with our hypothesis, we found that any hydrophobic residue at the 842 position conferred imatinib resistance (IC50 > 100nM), and all other amino acids tested to date were imatinib sensitive (IC50 < 100nM). Based on these data alone, over 55% of the 50 unique mutations identified from our PDGFRA-mutant GIST patient cohort would be predicted to be imatinib sensitive, demonstrating the importance of understanding the biochemical properties of various exon 18 mutations to predict treatment outcomes. Our predictive model would help identify patients with PDGFRA exon 18 mutations that could benefit from using front-line imatinib as an alternative to avapritinib, which has been recently approved for first-line treatment for all PDGFRA exon 18 mutant GIST. Imatinib, which has been used clinically for over 20 years, has a superior safety and tolerability profile compared to avapritinib. This paradigm shifting approach has the potential to transform patient care and outcomes by optimizing the sequencing of TKIs for PDGFRA-mutant GIST patients. Citation Format: Homma M. Khosroyani, Lillian R. Klug, Johanna Falkenhorst, Sebastian Bauer, Michael C. Heinrich. Predicting imatinib responses in exon 18 PDGFRA-mutant GIST [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 660.

Gastrointestinal stromal tumors (GIST) harboring activating mutations of PDGFRA respond to imatinib, with the notable exception of the most common mutation, D842V. Avapritinib is a novel, potent KIT/PDGFRA inhibitor with substantial clinical activity in patients with the D842V genotype. To date, only a minority of PDGFRA-mutant patients treated with avapritinib have developed secondary resistance. Tumor and plasma biopsies in 6 of 7 patients with PDGFRA primary mutations who progressed on avapritinib or imatinib had secondary resistance mutations within PDGFRA exons 13, 14, and 15 that interfere with avapritinib binding. Secondary PDGFRA mutations causing V658A, N659K, Y676C, and G680R substitutions were found in 2 or more patients each, representing recurrent mechanisms of PDGFRA GIST drug resistance. Notably, most PDGFRA-mutant GISTs refractory to avapritinib remain dependent on the PDGFRA oncogenic signal. Inhibitors that target PDGFRA protein stability or inhibition of PDGFRA-dependent signaling pathways may overcome avapritinib resistance. Significance: Here, we provide the first description of avapritinib resistance mechanisms in PDGFRA-mutant GIST. This article is highlighted in the In This Issue feature, p. 1

Targeted therapies for gastrointestinal stromal tumor (GIST) are modestly effective, but GIST cannot be cured with single agent tyrosine kinase inhibitors. In this study, we sought to identify new therapeutic targets in GIST by investigating the tumor microenvironment. Here, we identified a paracrine signaling network by which cancer-associated fibroblasts (CAFs) drive GIST growth and metastasis. Specifically, CAFs isolated from human tumors were found to produce high levels of platelet-derived growth factor C (PDGFC), which activated PDGFC-PDGFRA signal transduction in GIST cells that regulated the expression of SLUG, an epithelial-mesenchymal transition (EMT) transcription factor and downstream target of PDGFRA signaling. Together, this paracrine induce signal transduction cascade promoted tumor growth and metastasis in vivo. Moreover, in metastatic GIST patients, SLUG expression positively correlated with tumor size and mitotic index. Given that CAF paracrine signaling modulated GIST biology, we directly targeted CAFs with a dual PI3K/mTOR inhibitor, which synergized with imatinib to increase tumor cell killing and in vivo disease response. Taken together, we identified a previously unappreciated cellular target for GIST therapy in order to improve disease control and cure rates.

Mutations within the activation loop of members of the class III receptor tyrosine kinase (RTK) subfamily, which includes KIT, PDGFRA, and FLT3, have been observed in multiple human tumor types. These mutations confer constitutive activation as well as resistance to the type II tyrosine kinase inhibitors (TKI) that are currently clinically available, such as imatinib and sunitinib. It is now understood that activation loop mutations in class III RTKs shift the activation state equilibrium away from inactive states, to which type II TKIs bind, to the active state by destabilizing the inactive conformation. Recently, type I TKIs, which can bind to active kinase conformations, have been developed with specificity for class III RTK members. Some type I TKIs, such as crenolanib and avapritinib (BLU-285), have entered clinical studies for patients with activation loop mutations in KIT, PDGFRA, or FLT3. Preliminary results suggest that these type I TKIs show activity in these patient populations that previously lacked effective treatments. This article reviews the inactive and active structures of KIT, PDGFRA, and FLT3, how the mutations seen in human cancers affect kinase structure, and the clinical implications of these mutations in terms of type I vs. type II TKI binding.

Certain cancers, including gastrointestinal stromal tumor (GIST) and subsets of melanoma, are caused by somatic KIT mutations that result in KIT receptor tyrosine kinase constitutive activity, which drives proliferation. The treatment of KIT-mutant GIST has been revolutionized with the advent of KIT-directed cancer therapies. KIT tyrosine kinase inhibitors (TKI) are superior to conventional chemotherapy in their ability to control advanced KIT-mutant disease. However, these therapies have a limited duration of activity due to drug-resistant secondary KIT mutations that arise (or that are selected for) during KIT TKI treatment. To overcome the problem of KIT TKI resistance, we sought to identify novel therapeutic targets in KIT-mutant GIST and melanoma cells using a human tyrosine kinome siRNA screen. From this screen, we identified lemur tyrosine kinase 3 (LMTK3) and herein describe its role as a novel KIT regulator in KIT-mutant GIST and melanoma cells. We find that LMTK3 regulated the translation rate of KIT, such that loss of LMTK3 reduced total KIT, and thus KIT downstream signaling in cancer cells. Silencing of LMTK3 decreased cell viability and increased cell death in KIT-dependent, but not KIT-independent GIST and melanoma cell lines. Notably, LMTK3 silencing reduced viability of all KIT-mutant cell lines tested, even those with drug-resistant KIT secondary mutations. Furthermore, targeting of LMTK3 with siRNA delayed KIT-dependent GIST growth in a xenograft model. Our data suggest the potential of LMTK3 as a target for treatment of patients with KIT-mutant cancer, particularly after failure of KIT TKIs.

The diagnosis and treatment of gastrointestinal stromal tumor (GIST) has emerged as a paradigm for modern cancer treatment ('precision medicine'), as it highlights the importance of matching molecular defects with specific therapies. Over the past two decades, the molecular classification and diagnostic work up of GIST has been radically transformed, accompanied by the development of molecular therapies for specific subgroups of GIST. This review summarizes the developments in the field of molecular diagnosis of GIST, particularly as they relate to optimizing medical therapy. Areas covered: Based on an extensive literature search of the molecular and clinical aspects of GIST, the authors review the most important developments in this field with an emphasis on the differential diagnosis of GIST including mutation testing, therapeutic implications of each molecular subtype, and emerging technologies relevant to the field. Expert commentary: The use of molecular diagnostics to classify GIST has been shown to be successful in optimizing patient treatment, but these methods remain under-utilized. In order to facilitate efficient and comprehensive molecular testing, the authors have developed a decision tree to aid clinicians.

The functional inactivation of the metabolic enzyme complex succinate dehydrogenase (SDH) is a well-validated cause of multiple human cancers. Germline inheritance of a loss-of-function (LOF) SDHA mutation can result in SDH-deficiency is associated with a high lifetime risk of developing one or more SDH-deficient cancers. There are no highly effective medical therapies for advanced unresectable or metastatic SDH-deficient cancer, but most are curable if resected at an early stage. Early detection is therefore critical. However, despite knowing that loss of enzyme activity leads to tumor development, it has been difficult to determine which variants disrupt enzyme activity and contribute to cancer risk. There are currently over 600 SDHA missense reported in ClinVar, yet over 90% are considered variants of uncertain significance (VUS) due to a lack of functional evidence. Nearly 1% of the population carries a SDHA missense VUS and more are discovered every day with the increased availability of genetic testing modalities (clinical and direct-to-consumer). Only those carrying SDHA variant known to be LOF (pathogenic) can be recommended for enhanced tumor surveillance or genetic counseling. Currently, without an adequate model to determine the functional consequence of SDHA variants, an individual who carries SDHA VUS may have a significant lifetime risk of cancer, but are unlikely to benefit from early detection. In order to address this critical need, we sought to develop a novel human cell line model of SDHA-deficiency that would allow us to interrogate the functional consequences of SDHA VUS. Using CRISPR-Cas9 technology, we created a clonal HAP1 SDHA knockout cell line. Genetic inactivation of SDHA was verified by sequencing and immunoblotting, and resulted in complete loss of SDH activity. To investigate the utility of this model for characterizing the functional consequences of SDHA variants, we performed complementation experiments with SDHA variants with known functional consequences by lentiviral transduction. SDHA expression was confirmed by quantitative RT-PCR and immunoblotting and SDH activity was measured from isolated mitochondria using a commercial kit. As expected, expression of wild-type SDHA or known no-effect (benign) SDHA variants restores SDH-activity, whereas expression of known LOF (pathogenic) SDHA variants fails to restore enzyme activity. Using this model, we can functionally characterize SDHA VUS and obtain the evidence needed to reclassify their clinical significance. This model will lead to the discovery of novel pathogenic SDHA variants and help identify those at-risk for developing SDH-deficient cancers. Citation Format: Jason Kent, Lillian Klug, Michael Heinrich. A novel model for functional profiling of germline SDHA variants associated with cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 812.

Purpose: Patients who inherit a pathogenic loss-of-function genetic variant involving one of the four succinate dehydrogenase (SDH) subunit genes have up to an 86% chance of developing one or more cancers by the age of 50. If tumors are identified and removed early in these high-risk patients, they have a higher potential for cure. Unfortunately, many alterations identified in these genes are variants of unknown significance (VUS), confounding the identification of high-risk patients. If we could identify misclassified SDH VUS as benign or pathogenic SDH mutations, we could better select patients for cancer screening procedures and remove tumors at earlier stages. Experimental Design: In this study, we combine data from clinical observations, a functional yeast model, and a computational model to determine the pathogenicity of 22 SDHA VUS. We gathered SDHA VUS from two primary sources: The OHSU Knight Diagnostics Laboratory and the literature. We used a yeast model to identify the functional effect of a VUS on mitochondrial function with a variety of biochemical assays. The computational model was used to visualize variants' effect on protein structure. Results: We were able to draw conclusions on functional effects of variants using our three-prong approach to understanding VUS. We determined that 16 (73%) of the alterations are actually pathogenic, causing loss of SDH function, and six (27%) have no effect upon SDH function. Conclusions: We thus report the reclassification of the majority of the VUS tested as pathogenic, and highlight the need for more thorough functional assessment of inherited SDH variants. Clin Cancer Res; 23(21); 6733–43. ©2017 AACR.

Resistant KIT mutations have hindered the development of KIT kinase inhibitors for treatment of patients with systemic mastocytosis. The goal of this research was to characterize the synergistic effects of a novel combination therapy involving inhibition of KIT and calcineurin phosphatase, a nuclear factor of activated T cells (NFAT) regulator, using a panel of KIT-mutant mast cell lines. The effects of monotherapy or combination therapy on the cellular viability/survival of KIT-mutant mast cells were evaluated. In addition, NFAT-dependent transcriptional activity was monitored in a representative cell line to evaluate the mechanisms responsible for the efficacy of combination therapy. Finally, shRNA was used to stably knockdown calcineurin expression to confirm the role of calcineurin in the observed synergy. The combination of a KIT inhibitor and a calcineurin phosphatase inhibitor (CNPI) synergized to reduce cell viability and induce apoptosis in six distinct KIT-mutant mast cell lines. Both KIT inhibitors and CNPIs were found to decrease NFAT-dependent transcriptional activity. NFAT-specific inhibitors induced similar synergistic apoptosis induction as CNPIs when combined with a KIT inhibitor. Notably, NFAT was constitutively active in each KIT-mutant cell line tested. Knockdown of calcineurin subunit PPP3R1 sensitized cells to KIT inhibition and increased NFAT phosphorylation and cytoplasmic localization. Constitutive activation of NFAT appears to represent a novel and targetable characteristic of KIT-mutant mast cell disease. Our studies suggest that combining KIT inhibition with NFAT inhibition might represent a new treatment strategy for mast cell disease. Mol Cancer Ther; 13(12); 2840–51. ©2014 AACR.

e22512 Background: Activating PDGFRA mutations are seen in about 10% of gastrointestinal stromal tumors (GIST). Of these, the majority are one specific PDGFRA mutation, D842V, which confers resistance to all clinically approved tyrosine kinase inhibitors (TKI). Much has been learned about the mechanism by which TKIs work in GIST using cell lines derived from human GIST that bear activating KIT mutations. However, despite enormous effort to develop cell lines derived from human tumors over the past 15 years, there are currently no human cell models of PDGFRA D842V GIST. With the development of potent PDGFRA D842V TKI, such as crenolanib, avapritinib, and DCC2618 it is of interest to understand how these drugs affect PDGFRA-mutant GIST biology. Methods: We sought to convert the KIT-driven GIST-T1 cell line to be PDGFRA-driven by transduction with PDGFRA D842V. Endogenous KIT expression was knocked-out using CRISPR Cas9. Knockout was verified at protein and DNA levels. Clonal KIT knockout cell lines were tested for sensitivity to KIT and PDGFRA TKI both in vitro and in vivo models. Kinase dependence was determined by measuring the effect on viability after KIT and/or PDGFRA knockdown by siRNA. Results: GIST-T1 cells transduced with PDGFRA D842V display highly phosphorylated PDGFRA, imatinib resistance, and PDGFRA dependence. Cell lines that express both endogenous mutant KIT and exogenous PDGFRA D842V maintain some dependence on KIT, but less so than the parental KIT-driven GIST-T1 cell line. Complete knockout of KIT protein expression was achieved in GIST-T1 D842V cells using CRISPR-Cas9. GIST-T1 D842V KITko cell lines demonstrate sensitivity to TKI avapritinib, crenolanib, and DCC2618. Xenografts were established using GIST-T1 D842V KITko cell lines and are sensitive to avapritinib in vivo. Conclusions: The GIST-T1 D842V KITko cell line demonstrates ability to switch kinase allegiance resulting in PDGFRA-dependence and KIT-independence. This GIST-T1 D842V KITko cell line represents a novel model of PDGFRA-mutant GIST and can be used to understand the unique biological aspects of these tumors, particularly the response to new potent PDGFRA kinase inhibitors such as avapritinib which will have immense clinical relevance.

11044 Background: Multiple cancers, such as gastrointestinal stromal tumors (GIST) and melanoma, have been shown to be caused by somatic activating mutations in the receptor tyrosine kinase KIT. The major cause of death in patients with advanced KIT -mutant cancers is due to the development of KIT tyrosine kinase inhibitor-resistant (TKI-resistant) metastatic disease. Drug resistance arises almost exclusively from secondary mutations within KIT, highlighting the importance of KIT in the proliferation and survival of these tumors. Methods: We performed a human kinase siRNA screen in multiple KIT -mutant cancer cell lines using viability as a read out. We defined candidate targets as those whose knockdown decreased viability in all cell lines. Validation and mechanistic studies were done using a library of KIT-mutant GIST and melanoma cells. Results: We identified lemur tyrosine kinase 3 (LMTK3) as candidate target in three KIT -mutant cell lines. LMTK3 silencing reduced the viability of all KIT -mutant GIST and melanoma cells tested to date, including cell lines with KIT TKI-resistance mutations. Importantly, LMTK3 silencing decreased the viability of KIT -mutant cells specifically, but not that of KIT-independent GIST and melanoma cells. Further, we found that decreased cell viability was due to induction of apoptosis, as assessed by measuring caspase 3 and 7 activity within 96 hours of LMTK3 silencing. LMTK3 knockdown also reduced tumor growth in vivo in a GIST xenograft model. Because these cells depend so heavily on KIT and the loss of KIT signaling results in cell death, we hypothesized that LMTK3 silencing may affect this pathway. Indeed, LMTK3 silencing decreased levels of autophosphorylated KIT. We also observed a significant decrease in total KIT protein expression. This phenotype and corresponding viability was rescued with exogenous expression of full length LMTK3. Conclusions: LMTK3 is an important regulator of oncogenic KIT expression and activity in KIT-mutant GIST and melanoma and represents a novel, tractable target.

Lartruvo (olaratumab) is a monoclonal antibody against the extracellular domain of PDGFRA. Olaratumab blocks ligand binding and thereby inhibits activation of PDGFRA kinase activity. Pre-clinically, this antibody inhibited PDGFRA-dependent tumor growth. In a randomized Phase II study, adding olaratumab to doxorubicin chemotherapy significantly improved overall survival, leading to FDA approval.

BACKGROUND: Neuronal polarization is an essential step of morphogenesis and connectivity in the developing brain. The serine/threonine kinase LKB1 is a key regulator of cell polarity, metabolism, tumorigenesis, and is required for axon formation. It is allosterically regulated by two related and evolutionarily conserved pseudokinases, STe20-Related ADapters (STRADs) α and β. The roles of STRADα and STRADβ in the developing nervous system are not fully defined, nor is it known whether they serve distinct functions. RESULTS: We find that STRADα is highly spliced and appears to be the primal STRAD paralog. We report that each STRAD is sufficient for axogenesis and promoting cell survival in the developing cortex. We also reveal a reciprocal protein-stabilizing relationship in vivo between LKB1 and STRADα, whereby STRADα specifically maintains LKB1 protein levels via cytoplasmic compartmentalization. CONCLUSIONS: We demonstrate a novel role for STRADβ in axogenesis and also show for the first time in vivo that STRADα, but not STRADβ, is responsible for LKB1 protein stability.

Multiple cancers, such as gastrointestinal stromal tumors (GIST) and melanoma, have been shown to be caused by somatic activating mutations in the receptor tyrosine kinase KIT. The major cause of death in patients with advanced KIT-mutant cancers is due to KIT tyrosine kinase inhibitor-resistant metastatic disease. Resistance develops almost exclusively because of secondary mutations within KIT, highlighting the importance of KIT in the proliferation and survival of these tumors. Therefore, strategies that affect KIT protein expression and maturation may be therapeutic. In order to identify regulators of KIT that may represent novel targets, we performed an siRNA screen in multiple KIT-mutant cancer cell lines. From these screens we identified lemur tyrosine kinase 3 (LMTK3) as a novel KIT regulator and potential target in KIT-mutant cancers. LMTK3 has been implicated as an important protein in multiple cancers, including leukemia, breast, and colon cancer. However, the oncogenic function of LMTK3 is not entirely clear. Despite its name, lemur tyrosine kinase 3 is, in fact, a serine/ threonine kinase. Beyond its enzymatic kinase function, LMTK3 has also been shown to have important scaffolding functions. LMTK3 has not been previously known to interact with KIT, but does regulate the stability of other receptors. We have found that LMTK3 silencing reduced the viability of all KIT-mutant GIST and melanoma cells tested to date, including those with ATP binding pocket and activation loop resistance mutations. Importantly, LMTK3 silencing decreased the viability of KIT-mutant cells specifically, as silencing of LMTK3 in cells lacking KIT mutations did not affect their viability. Further, we found the decrease in cell viability was due to induction of apoptosis, as seen by cleavage of PARP within 96 hours of LMTK3 silencing. Because these cells depend so heavily on KIT and loss of KIT signaling results in cell death, we hypothesized that LMTK3 silencing may be affecting this pathway. Indeed, with LMTK3 silencing there was a significant decrease in phosphorylated KIT (Y719), which was accompanied by decreased phospho-AKT (S473). We also observed a robust decrease in total KIT protein levels. However, there was no decrease in KIT transcript levels, suggesting that LMTK3 plays a role in regulating KIT at the protein level. While other KIT protein regulators, such as HSP90, have been suggested as therapeutic targets for GIST, the ubiquitous functions of these targets makes these treatments sub-optimal, often causing toxicity. Silencing of LMTK3, on the other hand, specifically kills KIT-mutant cancer cells. Furthermore, as a kinase, inhibition of LMTK3 by a small molecule may be possible, and we are in the process of identifying LMTK3 inhibitors. LMTK3 is a positive regulator of oncogenic KIT in GIST and melanoma and represents a novel, tractable target in KIT-mutant cancers. Citation Format: Lillian R. Klug, Jeffery W. Tyner, Michael C. Heinrich. LMTK3 is a novel regulator of oncogenic KIT in KIT-mutant cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 191.