Supplementary Tables S1-S2, Figures S1-S9, and Methods Table S1: Raw siRNA screening results Figure S1: Response of GIST cell lines to the KIT inhibitor, imatinib Figure S2: Testing of individual FGFR3 siRNA duplexes for efficacy of reducing GIST T1 and GIST 10R cell viability Figure S3: Potential degradation product of FGFR3 upon inhibition with a small-molecule inhibitor Figure S4: FGF1 restores KIT phosphorylation and rescues GIST cells from imatinib inhibition Figure S5: SCF rescues GIST cells from FGFR inhibition Figure S6: FGFR expression levels after siRNA knockdown Figure S7: Combination of B-RAF inhibitor with imatinib is synergistic for GIST 10R cells Figure S8: FGF2 expression levels after siRNA knockdown Figure S9: Illustration of patient tissue analysis using the Aperio ScanScope CS Slide Scanner Supplemental Materials and Methods

The majority of gastrointestinal stromal tumors (GIST) harbor constitutively activating mutations in KIT tyrosine kinase. Imatinib, sunitinib, and regorafenib are available as first-, second-, and third-line targeted therapies, respectively, for metastatic or unresectable KIT-driven GIST. Treatment of patients with GIST with KIT kinase inhibitors generally leads to a partial response or stable disease but most patients eventually progress by developing secondary resistance mutations in KIT. Tumor heterogeneity for secondary resistant KIT mutations within the same patient adds further complexity to GIST treatment. Several other mechanisms converge and reactivate the MAPK pathway upon KIT/PDGFRA-targeted inhibition, generating treatment adaptation and impairing cytotoxicity. To address the multiple potential pathways of drug resistance in GIST, the KIT/PDGFRA inhibitor ripretinib was combined with MEK inhibitors in cell lines and mouse models. Ripretinib potently inhibits a broad spectrum of primary and drug-resistant KIT/PDGFRA mutants and is approved by the FDA for the treatment of adult patients with advanced GIST who have received previous treatment with 3 or more kinase inhibitors, including imatinib. Here we show that ripretinib treatment in combination with MEK inhibitors is effective at inducing and enhancing the apoptotic response and preventing growth of resistant colonies in both imatinib-sensitive and -resistant GIST cell lines, even after long-term removal of drugs. The effect was also observed in systemic mastocytosis (SM) cells, wherein the primary drug-resistant KIT D816V is the driver mutation. Our results show that the combination of KIT and MEK inhibition has the potential to induce cytocidal responses in GIST and SM cells.

Activating mutations in KIT/PDGFRA receptor tyrosine kinases drive gastrointestinal stromal tumors (GIST). KIT/PDGFRA inhibitors, such as imatinib do not evoke an effective cytocidal response, leaving room for quiescence and development of multiple secondary resistance mutations. As the majority of the secondary resistance clones activate PI3K and MAPK pathways, we investigated whether combined targeting of KIT/PI3K/MAPK (KPM) pathways overcomes drug resistance and quiescence in GIST cells. We monitored the proliferation of imatinib-sensitive and-resistant GIST cell lines after treating them with various combinations of drugs to inhibit KPM pathways. Cytocidal response was evaluated through proliferation, apoptosis and colony outgrowth assays. Combined inhibition of KPM signaling pathways using a KPM inhibitor cocktail decreased the survival of drug-resistant GIST cells and dramatically reduced their proliferation. Downstream pathway analysis showed that the residual PI3K/MAPK signaling observed after KIT inhibitor treatment plays a role in mediating quiescence and drug resistance. The KPM inhibitor cocktail with sunitinib or regorafenib effectively induced apoptosis and prevented colony outgrowth after long-term drug removal, suggesting that it can be used as an effective strategy against quiescence and drug resistance in metastatic GIST.

Abstract Background: Cancer cells activate autophagy, a catabolic process to resupply nutrients and recycle damaged organelles, in order to survive stresses such as limited nutrients and hypoxia, or chemotherapy treatments. RAS mutant cancers, in particular, have been found to require autophagy for tumor growth and survival.1,2 Treating RAS mutant tumors with inhibitors of the downstream MAPK pathway has been largely unsuccessful, as these drugs have been shown to further stimulate autophagy, allowing for tumor cell survival.3,4 Inhibiting autophagy in combination with MAPK pathway inhibition may represent a possible new treatment paradigm for RAS mutant cancers. Proof-of-concept for this strategy was obtained in cancer models and in a RAS mutant pancreatic cancer patient by blocking autophagy with derivatives of chloroquine, in combination with MAPK inhibitors.3,4 Chloroquines indirectly block autophagy via disruption of lysosomal function, which may also affect important normal cellular processes. Chloroquines accumulate in tissues, notably the brain, where autophagy may be vital for neuronal health. The potential exists to more selectively inhibit autophagy by targeting specific components of the autophagy pathway. ULK1/2 kinases initiate autophagy and provide the potential for a targeted approach for selectively inhibiting autophagy in RAS mutant cancers. Herein, we describe preclinical studies with the ULK inhibitor DCC-3116, designed as a potential inhibitor of autophagy in RAS mutant cancers. Methods: In vitro kinase assays were performed using cellular levels of ATP (1 mM) and a peptide substrate. In cell assays, ULK activity was assessed using an ELISA for phosphorylated ATG13. Autophagosome formation was measured using the dye, Cyto-ID. Autophagic flux was assessed using cells expressing the autophagy protein LC3 fused to luciferase. The synergy of DCC-3116 in combination with MAPK inhibitors was assessed in 2D or 3D cell growth assays. Xenograft models were used to assess pharmacokinetics (PK) and pharmacodynamics (PD), as well as efficacy in vivo. Results: DCC-3116 is a potent and selective inhibitor of ULK1/2, inhibiting no other kinases within 30-fold of ULK potency, and only 5 kinases within 100-fold. DCC-3116 inhibited phosphorylation of the ULK substrate ATG13 in cancer cell assays. DCC-3116 inhibited autophagosome formation, as well as degradation of the autophagy marker LC3. DCC-3116 exhibited synergy in vitro in combination with MAPK pathway inhibitors in inhibiting cancer cell growth. In PK/PD models, oral doses of DCC-3116 led to sustained inhibition of ATG13 phosphorylation. DCC-3116, in combination with MAPK inhibitors, exhibited additivity or synergy in inhibiting tumor growth in xenograft models. DCC-3116 exhibited low brain penetration in rats, minimizing inhibition of CNS autophagy. Conclusion: Selectively blocking autophagy via inhibition of ULK kinases, in combination with MAPK pathway inhibition, is a promising therapeutic approach for RAS mutant cancers. DCC-3116 warrants further study as an inhibitor of autophagy, and has been selected as a candidate for potential development in the treatment of RAS mutant cancers. Guo et al., Genes and Dev. 2011; 25: 460 Yang et al., Genes and Dev. 2011; 25: 717 Bryant et al., Nature Med. 2019; 25: 628 Kinsey et al., Nature Med. 2019; 25: 620 Citation Format: Bryan D Smith, Lakshminarayana Vogeti, Anu Gupta, Jarnail Singh, Gada Al-Ani, Stacie L Bulfer, Timothy M Caldwell, Mary J Timson, Subha Vogeti, Yu Mi Ahn, Hikmat Al-Hashimi, Chase K Crawley, Cale L Heiniger, Cynthia B Leary, Justin T Proto, Quanrong Shen, Hanumaiah Telikepalli, Karen Yates, Wei-Ping Lu, Daniel L Flynn. Preclinical studies with DCC-3116, an ULK kinase inhibitor designed to inhibit autophagy as a potential strategy to address mutant RAS cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B129. doi:10.1158/1535-7163.TARG-19-B129

Ripretinib (DCC-2618) was designed to inhibit the full spectrum of mutant KIT and PDGFRA kinases found in cancers and myeloproliferative neoplasms, particularly in gastrointestinal stromal tumors (GISTs), in which the heterogeneity of drug-resistant KIT mutations is a major challenge. Ripretinib is a “switch-control” kinase inhibitor that forces the activation loop (or activation “switch”) into an inactive conformation. Ripretinib inhibits all tested KIT and PDGFRA mutants, and notably is a type II kinase inhibitor demonstrated to broadly inhibit activation loop mutations in KIT and PDGFRA, previously thought only achievable with type I inhibitors. Ripretinib shows efficacy in preclinical cancer models, and preliminary clinical data provide proof-of-concept that ripretinib inhibits a wide range of KIT mutants in patients with drug-resistant GISTs.

Abstract Introduction: Activating mutations and other genetic alterations in KIT and PDGFRA receptor tyrosine kinases have been identified in certain cancers and proliferative diseases, including most cases of gastrointestinal stromal tumors (GIST) and systemic mastocytosis, and small percentages of gliomas, lung cancer, and leukemias. The treatment of metastatic GIST has been transformed with KIT inhibitors, but heterogeneous drug-resistant mutations arise during therapy, with individual patients often having multiple KIT mutations in different tumor sites. PDGFRA variants in GIST and other cancers also have a significant unmet medical need. DCC-2618 is a kinase switch control inhibitor that potently inhibits the spectrum of exon 9, 11, 13, 14, 17 and 18 mutations in KIT and exons 12, 14 and 18 mutations in PDGFRA. DCC-2618 has been designed to bind as a type II kinase inhibitor that forces the mutant kinases, including strongly activated mutants such as D816V KIT and D842V PDGFRA, into inactive conformations. DCC-2618 has been observed to be potent in enzyme and cell-based assays, and has demonstrated consistent efficacy in xenograft models driven by PDGFRA and KIT alterations. Methods: DCC-2618, and an active human metabolite, DP-5439, were tested for inhibition of PDGFRA and KIT mutants using standard enzyme and binding assays, and a variety of cell-based assays. Levels of phosphorylated PDGFRA and KIT were determined by Western blot or ELISA. Proliferation was measured using the fluorescent dye resazurin. An x-ray crystal structure of an analog of DCC-2618 was determined at Emerald Biostructures. The H1703 PDGFRA-amplified lung cancer and GIST T1 mutant KIT xenograft models were performed at MI Bioresearch. A GIST PDX exon 17 mutant KIT xenograft model was run at Molecular Response. Results: DCC-2618 and the metabolite DP-5439 inhibited KIT and PDGFRA variants with nanomolar potency. In CHO cells transfected with KIT or PDGFRA variants, DCC-2618 was shown to inhibit the full spectrum of the clinically relevant primary and refractory drug-resistant mutations tested. DCC-2618 also inhibited phosphorylation of KIT or PDGFRA in cell lines with various drug-resistant KIT mutations or PDGFRA alterations. DCC-2618 was compared to the FDA-approved KIT inhibitors imatinib, sunitinib, regorafenib, and midostaurin, as well as other KIT and PDGFRA inhibitors. In vivo, treatment with DCC-2618 led to tumor regressions in KIT- and PDGFRA-driven xenograft models. Conclusions: DCC-2618 has been observed to be a potent inhibitor of KIT and PDGFRA alterations, including mutants, fusions, and amplifications. Based on this profile, DCC-2618 may have utility in the treatment of KIT and PDGFRA-driven cancers including GIST, systemic mastocytosis, and a subset of lung cancers, gliomas, and leukemias. DCC-2618 is currently in a Phase 1 clinical trial in KIT and PDGFRA driven cancers (ClinicalTrials.gov Identifier: NCT02571036). Citation Format: Bryan D. Smith, Michael D. Kaufman, Anu Gupta, Cynthia B. Leary, Wei-ping Lu, Stacie L. Bulfer, Gada Al-Ani, Jarnail Singh, Subha Vogeti, Michael C. Heinrich, Daniel L. Flynn. Inhibition of oncogenic and drug-resistant PDGFRA and KIT alterations by DCC-2618 [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 3925.

Abstract Background: DCC-2618 is a potent switch control inhibitor of KIT and PDGFRα kinases maintains potent inhibition of mutant forms across all exon regions in preclinical models. Gastrointestinal stromal tumor (GIST) is an important disease to achieve a proof-of-concept due to the heterogeneity of resistance mutations in KIT which emerge on treatment with approved KIT inhibitors. In later lines of therapy resistance mechanisms independent of the KIT gene have also been described. Methods: The ongoing phase 1, PK-guided dose escalation study of DCC-2618 given orally BID [28-day cycle] tested doses from 20 mg to 200 mg in patients (pts) with advanced solid tumors including GIST (NCT02571036). We report preliminary longitudinal results of plasma cell free (cf) DNA sequencing by Guardant 360 collected throughout the study and levels of circulating tumor cells (CTCs) based on a viral telomerase promoter-driven GFP expression assay. Results: To date, 24 out of 31 enrolled pts had metastatic KITm GIST refractory to standard therapy. A high, total mean exposure of DCC-2618 and its active metabolite was achieved at 100 and 150 mg BID, affording steady state Cmax >5 µM in Cycle 1. Starting with 50 mg BID dose level, concentrations of total drug exceeding IC90 of the most resistant mutations to DCC-2618 were achieved. Next-generation sequencing of plasma cfDNA revealed a total of 40 KIT mutations in 16 of 18 GIST pts at baseline. DCC-2618 led to rapid decrease and/or clearance of the heterogeneous array of KIT mutations from plasma cfDNA including exons 9, 11, 13, 14, 17, and 18. Independent of suppressed KIT mutation burden, longitudinal monitoring of cfDNA revealed changes in non-KIT oncogenic mutations which may contribute to heterogenous mechanisms of resistance. KIT mutation burden will be correlated with metabolic response assessment by PET scans and exposure to DCC-2618. CTCs have been detected in blood from all GIST patients at baseline using a non-biased assay capable of identifying sarcoma cells. Preliminary result show that CTCs with immunofluorescent detection of KIT or PDGFRα, corresponding to their respective mutational status, show 1 of 3 patterns when compared to radiologic response: most pts show relatively stable low levels at stable disease (SD), a minority of pts with prolonged SD a decline in CTCs and 2 pts with progressive disease had significant increase in KIT positive CTCs. Conclusions: DCC-2618 and its active metabolite achieved high plasma concentrations sufficient to inhibit the most resistant KIT mutations at well-tolerated exposures. Monitoring of cfDNA KIT mutation frequency demonstrates rapid clearance of a broad spectrum of KIT mutations in this heavily pretreated GIST patient population and suggests candidate resistance genes that are independent of KIT. Our data provide a first signal that CTC monitoring might represent a potential marker for tumor control in KIT mutant GIST. Citation Format: Filip Janku, Albi Razak, Michael Gordon, David Brooks, Daniel Flynn, Anu Gupta, Michael Kaufman, Cynthia Leary, Bryan Smith, Deb Westwood, Neeta Somaiah, Elena Helman, Eric Gerstenberger, Oliver Rosen, Suzanne George. Translational research in a phase I proof-of-concept study supports that DCC-2618 is a pan-KIT inhibitor [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 LB-039. doi:10.1158/1538-7445.AM2017-LB-039

BACKGROUND & AIMS In gastrointestinal muscles, v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) is predominantly expressed by interstitial cells of Cajal (ICC) and platelet-derived growth factor receptor-α (PDGFRA) polypeptide is expressed by so-called fibroblast-like cells. KIT and PDGFRA have been reported to be coexpressed in ICC precursors and gastrointestinal stromal tumors (GISTs), which originate from the ICC lineage. PDGFRA signaling has been proposed to stimulate growth of GISTs that express mutant KIT, but the effects and mechanisms of selective blockade of PDGFRA are unclear. We investigated whether inhibiting PDGFRA could reduce proliferation of GIST cells with mutant KIT via effects on the KIT-dependent transcription factor ETV1. METHODS We studied 53 gastric, small intestinal, rectal, or abdominal GISTs collected immediately after surgery or archived as fixed blocks at the Mayo Clinic and University of California, San Diego. In human GIST cells carrying imatinib-sensitive and imatinib-resistant mutations in KIT, PDGFRA was reduced by RNA interference (knockdown) or inhibited with crenolanib besylate (a selective inhibitor of PDGFRA and PDGFRB). Mouse ICC precursors were retrovirally transduced to overexpress wild-type Kit. Cell proliferation was analyzed by methyltetrazolium, 5-ethynyl-2'-deoxyuridine incorporation, and Ki-67 immunofluorescence assays; we also analyzed growth of xenograft tumors in mice. Gastric ICC and ICC precursors, and their PDGFRA(+) subsets, were analyzed by flow cytometry and immunohistochemistry in wild-type, Kit(+/copGFP), Pdgfra(+/eGFP), and NOD/ShiLtJ mice. Immunoblots were used to quantify protein expression and phosphorylation. RESULTS KIT and PDGFRA were coexpressed in 3%-5% of mouse ICC, 35%-44% of ICC precursors, and most human GIST samples and cell lines. PDGFRA knockdown or inhibition with crenolanib efficiently reduced proliferation of imatinib-sensitive and imatinib-resistant KIT(+)ETV1(+)PDGFRA(+) GIST cells (50% maximal inhibitory concentration = 5-32 nM), but not of cells lacking KIT, ETV1, or PDGFRA (50% maximal inhibitory concentration >230 nM). Crenolanib inhibited phosphorylation of PDGFRA and PDGFRB, but not KIT. However, Kit overexpression sensitized mouse ICC precursors to crenolanib. ETV1 knockdown reduced KIT expression and GIST proliferation. Crenolanib down-regulated ETV1 by inhibiting extracellular-signal-regulated kinase (ERK)-dependent stabilization of ETV1 protein and also reduced expression of KIT and PDGFRA. CONCLUSIONS In KIT-mutant GIST, inhibition of PDGFRA disrupts a KIT-ERK-ETV1-KIT signaling loop by inhibiting ERK activation. The PDGFRA inhibitor crenolanib might be used to treat patients with imatinib-resistant, KIT-mutant GIST.

Abstract Kinase inhibitors such as imatinib have dramatically improved outcomes for patients with gastrointestinal stromal tumor (GIST), but many patients develop resistance to these treatments. Although in some patients this event corresponds with mutations in the GIST driver oncogenic kinase KIT, other patients develop resistance without KIT mutations. In this study, we address this patient subset in reporting a functional dependence of GIST on the FGF receptor FGFR3 and its crosstalk with KIT in GIST cells. Addition of the FGFR3 ligand FGF2 to GIST cells restored KIT phosphorylation during imatinib treatment, allowing sensitive cells to proliferate in the presence of the drug. FGF2 expression was increased in imatinib-resistant GIST cells, the growth of which was blocked by RNAi-mediated silencing of FGFR3. Moreover, combining KIT and FGFR3 inhibitors synergized to block the growth of imatinib-resistant cells. Signaling crosstalk between KIT and FGFR3 activated the MAPK pathway to promote resistance to imatinib. Clinically, an IHC analysis of tumor specimens from imatinib-resistant GIST patients revealed a relative increase in FGF2 levels, with a trend toward increased expression in imatinib-naïve samples consistent with possible involvement in drug resistance. Our findings provide a mechanistic rationale to evaluate existing FGFR inhibitors and multikinase inhibitors that target FGFR3 as promising strategies to improve treatment of patients with GIST with de novo or acquired resistance to imatinib. Cancer Res; 75(5); 880–91. ©2015 AACR.

Stem cell factor (mouse: Kitl, human: KITLG) and insulin-like growth factor-1 (IGF1), acting via KIT and IGF1 receptor (IGF1R), respectively, are critical for the development and integrity of several tissues. Autocrine/paracrine KITLG-KIT and IGF1-IGF1R signaling are also activated in several cancers including gastrointestinal stromal tumors (GIST), the most common sarcoma. In murine gastric muscles, IGF1 promotes Kitl-dependent development of interstitial cells of Cajal (ICC), the non-neoplastic counterpart of GIST, suggesting cooperation between these pathways. Here, we report a novel mechanism linking IGF1-IGF1R and KITLG-KIT signaling in both normal and neoplastic cells. In murine gastric muscles, the microenvironment for ICC and GIST, human hepatic stellate cells (LX-2), a model for cancer niches, and GIST cells, IGF1 stimulated Kitl/KITLG protein and mRNA expression and promoter activity by activating several signaling pathways including AKT-mediated glycogen synthase kinase-3β inhibition (GSK3i). GSK3i alone also stimulated Kitl/KITLG expression without activating mitogenic pathways. Both IGF1 and GSK3i induced chromatin-level changes favoring transcriptional activation at the Kitl promoter including increased histone H3/H4 acetylation and H3 lysine (K) 4 methylation, reduced H3K9 and H3K27 methylation and reduced occupancy by the H3K27 methyltransferase EZH2. By pharmacological or RNA interference-mediated inhibition of chromatin modifiers we demonstrated that these changes have the predicted impact on KITLG expression. KITLG knock-down and immunoneutralization inhibited the proliferation of GIST cells expressing wild-type KIT, signifying oncogenic autocrine/paracrine KITLG-KIT signaling. We conclude that membrane-to-nucleus signaling involving GSK3i establishes a previously unrecognized link between the IGF1-IGF1R and KITLG-KIT pathways, which is active in both physiologic and oncogenic contexts and can be exploited for therapeutic purposes.

Mutations in the catalytic domain at the gatekeeper position represent the most prominent drug-resistant variants of kinases and significantly impair the efficacy of targeted cancer therapies. Understanding the mechanisms of drug resistance at the molecular and atomic levels will aid in the design and development of inhibitors that have the potential to overcome these resistance mutations. Herein, by introducing adaptive elements into the inhibitor core structure, we undertake the structure-based development of type II hybrid inhibitors to overcome gatekeeper drug-resistant mutations in cSrc-T338M, as well as clinically relevant tyrosine kinase KIT-T670I and Abl-T315I variants, as essential targets in gastrointestinal stromal tumors (GISTs) and chronic myelogenous leukemia (CML). Using protein X-ray crystallography, we confirm the anticipated binding mode in cSrc, which proved to be essential for overcoming the respective resistances. More importantly, the novel compounds effectively inhibit clinically relevant gatekeeper mutants of KIT and Abl in biochemical and cellular studies.

Although gastrointestinal stromal tumors (GISTs) harboring activating KIT or platelet-derived growth factor receptor A (PDGFRA) mutations respond to treatment with targeted KIT/PDGFRA inhibitors such as imatinib mesylate, these treatments are rarely curative. Most often, a sizeable tumor cell subpopulation survives and remains quiescent for years, eventually resulting in acquired resistance and treatment failure. Here, we report that imatinib induces autophagy as a survival pathway in quiescent GIST cells. Inhibiting autophagy, using RNAi-mediated silencing of autophagy regulators (ATGs) or antimalarial lysosomotrophic agents, promotes the death of GIST cells both in vitro and in vivo. Thus, combining imatinib with autophagy inhibition represents a potentially valuable strategy to promote GIST cytotoxicity and to diminish both cellular quiescence and acquired resistance in GIST patients.

Cellular retinoic acid binding protein II (CRABP-II) is overexpressed in a wide variety of cancers. Previously we have shown that CRABP-II expression levels are also elevated in neuroblastoma and Wilms tumors. To elucidate the molecular mechanisms underlying the abnormal expression of CRABP-II in Wilms tumor, we studied the expression of MycN and CRABP-II in these tumor samples. Our data revealed that CRABP-II is overexpressed in Wilms tumor compared to normal adjacent non-neoplastic tissue and its levels are even higher in late stage tumors. Its expression correlates with MycN expression in tumors. The tumors that do not express MycN have no CRABP-II expression. The expression of CRABP-II is also regulated by methylation and its promoter is unmethylated in tumors. Knockdown of MycN by small interfering RNA leads to downregulation of CRABP-II. Thus our results suggest that both MycN and DNA methylation are responsible for CRABP-II expression in pediatric tumors and demethylation of CRABP-II may be an early event in tumor development.

Glucocorticosteroid hormones, including prednisone and dexamethasone (Dex), have been used to treat lymphoid malignancies for many years because they readily induce apoptosis in immature lymphocytes lacking Bcl-2. However, elevated expression of the anti-apoptotic protein Bcl-2 inhibits apoptosis and contributes to glucocorticoid resistance. Using the Bcl-2-negative WEHI7.2 lymphoma line as an experimental model, we found that Dex not only induces apoptosis but also induces autophagy. The caspase inhibitor Z-VAD-fmk inhibited apoptosis but not autophagy in Dex-treated cells. Bcl-2 overexpression inhibited Dex-induced apoptosis even more potently than Z-VAD-fmk and, contrary to previous reports, Bcl-2 neither interacted with Beclin-1 nor inhibited autophagy. Rather, Bcl-2 overexpression facilitated detection of Dex-induced autophagy by both steady state methods and flux measurements, ostensibly due to apoptosis inhibition. Autophagy contributed to prolonged survival of Bcl-2-positive lymphoma cells following Dex treatment, as survival was reduced when autophagy was inhibited by 3-methyladenine. These findings emphasize the important interplay between apoptosis and autophagy and suggest a novel mechanism by which Bcl-2, which is frequently elevated in lymphoid malignancies, contributes to glucocorticoid resistance and survival of lymphoma cells.

Neuroblastoma is a heterogeneous disease in which 22% of tumors show MycN oncogene amplification and are associated with poor clinical outcome. MycN is a transcription factor that regulates the expression of a number of proteins that affect the clinical behavior of neuroblastoma. We report here that cellular retinoic acid-binding protein II (CRABP-II) is a novel MycN target, expressed at significantly higher levels in primary neuroblastoma tumors with mycN oncogene amplification as compared with non-MycN-amplified tumors. Moreover, regulated induction and repression of MycN in a neuroblastoma-derived cell line resulted in temporal and proportionate expression of CRABP-II. CRABP-II is expressed in several cancers, but its role in tumorigenesis has not been elucidated. We show that MycN binds to the promoter of CRABP-II and induces CRABP-II transcription directly. In addition, CRABP-II-transfected neuroblastoma cell lines show an increase in MycN protein levels resulting in increased cell motility. Gene expression profiling of CRABP-II-expressing cell lines uncovered increased expression of the HuB (Hel N1) gene. Hu proteins have been implicated in regulating the stability of MycN mRNA and other mRNAs by binding to their 3' untranslated regions. We did not, however, observe any change in MycN mRNA stability or protein half-life in response to CRABP-II expression. In contrast, de novo MycN protein synthesis was increased in CRABP-II-expressing neuroblastoma cells, thereby suggesting an autoregulatory loop that might exacerbate the effects of MycN gene amplification and affect the clinical outcome. Our findings also suggest that CRABP-II may be a potential therapeutic target for neuroblastoma.