Joseph D. Growney

Inactivating mutations of RNF43 confer Wnt dependency in pancreatic ductal adenocarcinoma

A growing number of agents targeting ligand-induced Wnt/β-catenin signaling are being developed for cancer therapy. However, clinical development of these molecules is challenging because of the lack of a genetic strategy to identify human tumors dependent on ligand-induced Wnt/β-catenin signaling. Ubiquitin E3 ligase ring finger 43 (RNF43) has been suggested as a negative regulator of Wnt signaling, and mutations of RNF43 have been identified in various tumors, including cystic pancreatic tumors. However, loss of function study of RNF43 in cell culture has not been conducted, and the functional significance of RNF43 mutations in cancer is unknown. Here, we show that RNF43 inhibits Wnt/β-catenin signaling by reducing the membrane level of Frizzled in pancreatic cancer cells, serving as a negative feedback mechanism. Inhibition of endogenous Wnt/β-catenin signaling increased the cell surface level of Frizzled. A panel of 39 pancreatic cancer cell lines was tested for Wnt dependency using LGK974, a selective Porcupine inhibitor being examined in a phase 1 clinical trial. Strikingly, all LGK974-sensitive lines carried inactivating mutations of RNF43. Inhibition of Wnt secretion, depletion of β-catenin, or expression of wild-type RNF43 blocked proliferation of RNF43 mutant but not RNF43–wild-type pancreatic cancer cells. LGK974 inhibited proliferation and induced differentiation of RNF43-mutant pancreatic adenocarcinoma xenograft models. Our data suggest that mutational inactivation of RNF43 in pancreatic adenocarcinoma confers Wnt dependency, and the presence of RNF43 mutations could be used as a predictive biomarker for patient selection supporting the clinical development of Wnt inhibitors in subtypes of cancer.

IDH1 mutations alter citric acid cycle metabolism and increase dependence on oxidative mitochondrial metabolism.

Oncogenic mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in several types of cancer, but the metabolic consequences of these genetic changes are not fully understood. In this study, we performed (13)C metabolic flux analysis on a panel of isogenic cell lines containing heterozygous IDH1/2 mutations. We observed that under hypoxic conditions, IDH1-mutant cells exhibited increased oxidative tricarboxylic acid metabolism along with decreased reductive glutamine metabolism, but not IDH2-mutant cells. However, selective inhibition of mutant IDH1 enzyme function could not reverse the defect in reductive carboxylation activity. Furthermore, this metabolic reprogramming increased the sensitivity of IDH1-mutant cells to hypoxia or electron transport chain inhibition in vitro. Lastly, IDH1-mutant cells also grew poorly as subcutaneous xenografts within a hypoxic in vivo microenvironment. Together, our results suggest therapeutic opportunities to exploit the metabolic vulnerabilities specific to IDH1 mutation.

Activity of deacetylase inhibitor panobinostat (LBH589) in cutaneous T-cell lymphoma models: Defining molecular mechanisms of resistance.

Panobinostat (LBH589) is a highly potent deacetylase inhibitor that has demonstrated clinical efficacy in patients with advanced cutaneous T‐cell lymphoma (CTCL). To gain a better understanding of the compound activity in this tumor type, we investigated the cellular and molecular effects of panobinostat using both in vitro and in vivo models of CTCL. All 4 tested CTCL cell lines exhibited very high sensitivity to panobinostat‐induced growth inhibition. However, only 2 of 4 lines exhibited significant response to the cytotoxic activity of panobinostat. In a CTCL xenograft mouse tumor model, panobinostat treatment resulted in complete tumor regression. The difference in cell sensitivity to panobinostat‐induced death enabled us to further investigate potential mechanisms responsible for tumor sensitivity or resistance. In CTCL cell lines that were insensitive to panobinostat‐induced apoptosis, constitutively activated NF‐κB and high levels of Bcl‐2 were observed. Inhibition of Bcl‐2 sensitized cells to the cytotoxic activity of panobinostat. Conversely, knockdown of Bax diminished the CTCL cell sensitivity. Interestingly, panobinostat could induce cytotoxicity in vorinostat‐resistant CTCL cells by downregulating phosphorylated STAT3 and STAT5 proteins. These studies suggest distinct mechanisms responsible for resistance to different deacetylase inhibitors. We show that the intrinsic apoptotic signaling plays an essential role in mediating panobinostat anticancer activity. Moreover, cancer cell sensitivity to panobinostat treatment may be further improved by combination with inhibition of anti‐apoptotic factors. These data provide preclinical support that panobinostat, as a single agent or in combination with other anticancer agents, is a promising therapy for CTCL.

Multivalent nanobodies targeting death receptor 5 elicit superior tumor cell killing through efficient caspase induction

Multiple therapeutic agonists of death receptor 5 (DR5) have been developed and are under clinical evaluation. Although these agonists demonstrate significant anti-tumor activity in preclinical models, the clinical efficacy in human cancer patients has been notably disappointing. One possible explanation might be that the current classes of therapeutic molecules are not sufficiently potent to elicit significant response in patients, particularly for dimeric antibody agonists that require secondary cross-linking via Fcγ receptors expressed on immune cells to achieve optimal clustering of DR5. To overcome this limitation, a novel multivalent Nanobody approach was taken with the goal of generating a significantly more potent DR5 agonist. In the present study, we show that trivalent DR5 targeting Nanobodies mimic the activity of natural ligand, and furthermore, increasing the valency of domains to tetramer and pentamer markedly increased potency of cell killing on tumor cells, with pentamers being more potent than tetramers in vitro. Increased potency was attributed to faster kinetics of death-inducing signaling complex assembly and caspase-8 and caspase-3 activation. In vivo, multivalent Nanobody molecules elicited superior anti-tumor activity compared to a conventional DR5 agonist antibody, including the ability to induce tumor regression in an insensitive patient-derived primary pancreatic tumor model. Furthermore, complete responses to Nanobody treatment were obtained in up to 50% of patient-derived primary pancreatic and colon tumor models, suggesting that multivalent DR5 Nanobodies may represent a significant new therapeutic modality for targeting death receptor signaling.

Gene Expression Ratios Lead to Accurate and Translatable Predictors of DR5 Agonism across Multiple Tumor Lineages

Death Receptor 5 (DR5) agonists demonstrate anti-tumor activity in preclinical models but have yet to demonstrate robust clinical responses. A key limitation may be the lack of patient selection strategies to identify those most likely to respond to treatment. To overcome this limitation, we screened a DR5 agonist Nanobody across >600 cell lines representing 21 tumor lineages and assessed molecular features associated with response. High expression of DR5 and Casp8 were significantly associated with sensitivity, but their expression thresholds were difficult to translate due to low dynamic ranges. To address the translational challenge of establishing thresholds of gene expression, we developed a classifier based on ratios of genes that predicted response across lineages. The ratio classifier outperformed the DR5+Casp8 classifier, as well as standard approaches for feature selection and classification using genes, instead of ratios. This classifier was independently validated using 11 primary patient-derived pancreatic xenograft models showing perfect predictions as well as a striking linearity between prediction probability and anti-tumor response. A network analysis of the genes in the ratio classifier captured important biological relationships mediating drug response, specifically identifying key positive and negative regulators of DR5 mediated apoptosis, including DR5, CASP8, BID, cFLIP, XIAP and PEA15. Importantly, the ratio classifier shows translatability across gene expression platforms (from Affymetrix microarrays to RNA-seq) and across model systems (in vitro to in vivo). Our approach of using gene expression ratios presents a robust and novel method for constructing translatable biomarkers of compound response, which can also probe the underlying biology of treatment response.

Abstract LB-139: IDH1 mutations alter citric acid cycle metabolism and increase dependence on oxidative mitochondrial metabolism

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Mutations in the genes encoding isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in a variety of tumor types, resulting in production of the proposed oncometabolite, 2-hydroxyglutarate (2-HG). How mutant IDH alters central carbon metabolism, though, remains unclear. To address this question, we performed 13C metabolic flux analysis (MFA) on an isogenic cell panel containing heterozygous IDH1/2 mutations. We observe a dramatic and consistent decrease in the ability of IDH1, but not IDH2, mutant cell lines to utilize reductive glutamine metabolism via the carboxylation of α-ketoglutarate to isocitrate. Additionally we find that cells with IDH1 mutations exhibit increased oxidative tricarboxylic acid (TCA) metabolism. Similar metabolic trends were observed in vivo as well, and also in endogenous, non-engineered IDH1/2 mutant cell lines. Interestingly, IDH1-mutant specific inhibitors were unable to reverse the decrease in reductive metabolism, suggesting that this metabolic phenotype is independent of 2-HG. Furthermore, this metabolic reprogramming increases the sensitivity of IDH1 mutant cells to hypoxia or electron transport chain (ETC) inhibition in vitro . IDH1 mutant cells also grow poorly as subcutaneous xenografts within hypoxic in vivo microenvironments. These results suggest that exploiting metabolic defects specific to IDH1 mutant cells could be an interesting avenue to explore therapeutically. Citation Format: Alexandra R. Grassian, Seth Parker, Shawn Davidson, Ajit Divakaruni, Courtney Green, Xiamei Zhang, Kelly Slocum, Minying Pu, Fallon Lin, Chad Vickers, Carol Joud-Caldwell, Franklin Chung, Hong Yin, Erika Handly, Christopher Straub, Joseph D. Growney, Matt Vander Heiden, Anne Murphy, Raymond Pagliarini, Christian Metallo. IDH1 mutations alter citric acid cycle metabolism and increase dependence on oxidative mitochondrial metabolism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-139. doi:10.1158/1538-7445.AM2014-LB-139

Abstract 2239: The effect combining the KIT inhibitor Imatinib with the PI3K inhibitor BKM120 or the dual PI3K/mTOR inhibitor BEZ235 on the proliferation of gastrointestinal stromal tumor cell lines

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Gastrointestinal stromal tumors (GIST) are the most common type of sarcoma found in the gastrointestinal tract, with 5,000-6,000 cases per year diagnosed in the United States. Activating mutations in KIT, or occasionally in PDGFR are present in up to 90% of GISTs. The PI3K/AKT pathway has been demonstrated to be a crucial survival pathway in imatinib-resistant GISTs. In this study, we evaluated single agent activities of the KIT inhibitor imatinib, the PI3K inhibitor BKM120 and the dual PI3K/mTOR inhibitor BEZ235, as well as the synergistic activity of the combination of imatinib with BKM120 and imatinib with BEZ235 in a panel of GIST cell lines. For example, when the antiproliferative effects of imatinib and BKM120 were evaluated in combination at concentrations from 0.37 µM to 3.3 µM, growth suppression was observed in excess of the percent inhibition achieved by imatinib or BKM120 single agent treatment in the GIST882 and GIST430 cell lines. Our results suggest that combining imatinib with BKM120 or BEZ235 may provide a therapeutic strategy for imatinib-resistant GISTs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2239. doi:1538-7445.AM2012-2239

Optimization of 3-Pyrimidin-4-yl-oxazolidin-2-ones as Orally Bioavailable and Brain Penetrant Mutant IDH1 Inhibitors

Mutant isocitrate dehydrogenase 1 (IDH1) is an attractive therapeutic target for the treatment of various cancers such as AML, glioma, and glioblastoma. We have evaluated 3-pyrimidin-4-yl-oxazolidin-2-ones as mutant IDH1 inhibitors that bind to an allosteric, induced pocket of IDH1R132H. This Letter describes SAR exploration focused on improving both the in vitro and in vivo metabolic stability of the compounds, leading to the identification of 19 as a potent and selective mutant IDH1 inhibitor that has demonstrated brain penetration and excellent oral bioavailability in rodents. In a preclinical patient-derived IDH1 mutant xenograft tumor model study, 19 efficiently inhibited the production of the biomarker 2-HG.

Abstract B65: Antitumor activity of imatinib in GIST is attenuated by FGFR signaling.

Activating mutations in either KIT or PDGFR are present in approximately 90% of gastrointestinal stromal tumors (GISTs). Treatment with the KIT and PDGFR inhibitor imatinib can control advanced disease in about 80% of GIST patients. However, the beneficial effect is not durable with more than 50% of patients progressing after 2 years of imatinib first line therapy. Gene expression data has revealed that FGF2 and FGFR1 are highly expressed in all primary GIST samples examined and at significantly higher levels than the other forty tumor types tested, suggesting that FGFR signaling may limit the antitumor response of imatinib. The effect of inhibiting FGFR signaling in GIST cell lines was tested by combining imatinib with BGJ398, a potent and selective small-molecule inhibitor of FGFR1, FGFR2, and FGFR3. This combination showed increased growth inhibition in imatinib-sensitive GIST cell lines. This effect was observed in the presence or absence of added FGF2. The degree of synergy generated by imatinib and BGJ398 combination correlated with the level of added FGF2. In addition, inhibition of mitogen-activated protein kinase (MAPK) was transient in GIST cells treated with imatinib. The re-activation of extracellular signal-regulated kinase (ERK) in GIST cells was accompanied by increased FGFR signaling, and can be suppressed by BGJ398 treatment. These results suggest that imatinib treatment induces feedback activation of FGFR signaling that can attenuate the antitumor effects of imatinib. This provides a rationale for combining imatinib and FGFR inhibitors, such as BGJ398, in the first line therapy of GIST. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B65. Citation Format: Fang Li, Joseph Growney, Linda Battalagine, Shumei Qiu, Paul Manley, Robert Schlegel, John Monahan. Antitumor activity of imatinib in GIST is attenuated by FGFR signaling. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B65.

Abstract 1620: Dovitinib has anti-tumor activity in gastrointestinal stromal tumor (GIST) cell lines.

Background: Gastrointestinal stromal tumors (GISTs) are the most common gastrointestinal tract sarcoma, with 5000-6000 cases per year diagnosed in the United States. Most GISTs carry activating mutations in KIT (≈ 75% of cases) or PDGFR alpha (10%-15%). Imatinib, a KIT/PDGFR inhibitor, is the frontline treatment for GIST, although secondary mutations commonly lead to acquired resistance. Dovitinib inhibits several kinase targets important in the development and maintenance of GISTs, including KIT, PDGFR, VEGFR, and FGFR. This study was designed to evaluate the single-agent activities of imatinib and dovitinib in a panel of GIST cell lines and an imatinib-sensitive mouse model. Methods: In vitro sensitivity was examined in both imatinib-sensitive (GIST-T1 and GIST882) and -insensitive (GIST430 and GIST48) cell lines. KIT phosphorylation was measured by Western blot and sandwich ELISA. Imatinib and dovitinib monotherapy were evaluated for tumor growth inhibition and delay in a nu/nu mouse xenograft model using GIST-T1 xenografts. Dovitinib was administered by mouth at either 60 mg/kg daily for 14 days or 30 mg/kg daily for 21 days. Imatinib was given at 100 mg/kg by mouth twice daily. Following treatment, dosing was terminated, and tumors were allowed to regrow off treatment. Kaplan-Meier analysis was used to determine tumor growth delay, reported as median time to reach endpoint (tumor volume ≥ 1000 mm 3 ). Results: Imatinib and dovitinib inhibit KIT phosphorylation in GIST-T1, GIST882, GIST430 and GIST48 cells, although imatinib appeared to be more potent. Dovitinib and imatinib both potently inhibited the proliferation, GI 50 10-100 nM, of imatinib-sensitive GIST-T1 and GIST882 cell lines; GI 50 was > 500 nM in imatinib-insensitive GIST48 and GIST430 cell lines. In vivo, imatinib and dovitinib were well tolerated for 14- and 21-day treatment cycles. In the 14-day treatment model, imatinib and dovitinib led to 52% and 59% tumor regression, respectively, and median time to regrowth was 69 days for both agents compared with 31 days for vehicle. In the 21-day treatment model, imatinib and dovitinib led to 66% and 36% tumor regression, respectively, and median time to regrowth was 81 and 69 days, respectively, compared with 27 days for vehicle. Conclusions: Dovitinib inhibits KIT signaling and has similar growth-inhibition activity to imatinib in imatinib-sensitive GIST cell lines. In xenograft models, both imatinib and dovitinib are well tolerated, induce tumor regression, and delay tumor regrowth. The additional kinase inhibitory activities of dovitinib offer the possibility for enhanced or differentiated activity to imatinib in certain settings. Experiments are ongoing to explore this possibility. These data suggest that dovitinib may have therapeutic benefit in GIST, with efficacy resulting from a combination of the inhibition of KIT and other kinases. Citation Format: Joseph D. Growney, Fang Li, Shumei Qiu, Bella Gorbatcheva, Linda Battalagine, Juergen Mestan, Paul Manley, Matthew Squires, Alex Cao, John E. Monahan. Dovitinib has anti-tumor activity in gastrointestinal stromal tumor (GIST) cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1620. doi:10.1158/1538-7445.AM2013-1620