Christopher Winter

Inhibition of NOTCH signaling by gamma secretase inhibitor engages the RB pathway and elicits cell cycle exit in T-cell acute lymphoblastic leukemia cells.

NOTCH signaling is deregulated in the majority of T-cell acute lymphoblastic leukemias (T-ALL) as a result of activating mutations in NOTCH1. Gamma secretase inhibitors (GSI) block proteolytic activation of NOTCH receptors and may provide a targeted therapy for T-ALL. We have investigated the mechanisms of GSI sensitivity across a panel of T-ALL cell lines, yielding an approach for patient stratification based on pathway activity and also providing a rational combination strategy for enhanced response to GSI. Whereas the NOTCH1 mutation status does not serve as a predictor of GSI sensitivity, a gene expression signature of NOTCH pathway activity does correlate with response, and may be useful in the selection of patients more likely to respond to GSI. Furthermore, inhibition of the NOTCH pathway activity signature correlates with the induction of the cyclin-dependent kinase inhibitors CDKN2D (p19(INK4d)) and CDKN1B (p27(Kip1)), leading to derepression of RB and subsequent exit from the cell cycle. Consistent with this evidence of cell cycle exit, short-term exposure of GSI resulted in sustained molecular and phenotypic effects after withdrawal of the compound. Combination treatment with GSI and a small molecule inhibitor of CDK4 produced synergistic growth inhibition, providing evidence that GSI engagement of the CDK4/RB pathway is an important mechanism of GSI action and supports further investigation of this combination for improved efficacy in treating T-ALL.

First Selective Small Molecule Inhibitor of FGFR4 for the Treatment of Hepatocellular Carcinomas with an Activated FGFR4 Signaling Pathway

UNLABELLED Aberrant signaling through the fibroblast growth factor 19 (FGF19)/fibroblast growth factor receptor 4 (FGFR 4) signaling complex has been shown to cause hepatocellular carcinoma (HCC) in mice and has been implicated to play a similar role in humans. We have developed BLU9931, a potent and irreversible small-molecule inhibitor of FGFR4, as a targeted therapy to treat patients with HCC whose tumors have an activated FGFR4 signaling pathway. BLU9931 is exquisitely selective for FGFR4 versus other FGFR family members and all other kinases. BLU9931 shows remarkable antitumor activity in mice bearing an HCC tumor xenograft that overexpresses FGF19 due to amplification as well as a liver tumor xenograft that overexpresses FGF19 mRNA but lacks FGF19 amplification. Approximately one third of patients with HCC whose tumors express FGF19 together with FGFR4 and its coreceptor klotho β (KLB) could potentially respond to treatment with an FGFR4 inhibitor. These findings are the first demonstration of a therapeutic strategy that targets a subset of patients with HCC. SIGNIFICANCE This article documents the discovery of BLU9931, a novel irreversible kinase inhibitor that specifically targets FGFR4 while sparing all other FGFR paralogs and demonstrates exquisite kinome selectivity. BLU9931 is efficacious in tumors with an intact FGFR4 signaling pathway that includes FGF19, FGFR4, and KLB. BLU9931 is the first FGFR4-selective molecule for the treatment of patients with HCC with aberrant FGFR4 signaling.

Interconnecting molecular pathways in the pathogenesis and drug sensitivity of T-cell acute lymphoblastic leukemia

To identify dysregulated pathways in distinct phases of NOTCH1-mediated T-cell leukemogenesis, as well as small-molecule inhibitors that could synergize with or substitute for gamma-secretase inhibitors (GSIs) in T-cell acute lymphoblastic leukemia (T-ALL) therapy, we compared gene expression profiles in a Notch1-induced mouse model of T-ALL with those in human T-ALL. The overall patterns of NOTCH1-mediated gene expression in human and mouse T-ALLs were remarkably similar, as defined early in transformation in the mouse by the regulation of MYC and its target genes and activation of nuclear factor-kappaB and PI3K/AKT pathways. Later events in murine Notch1-mediated leukemogenesis included down-regulation of genes encoding tumor suppressors and negative cell cycle regulators. Gene set enrichment analysis and connectivity map algorithm predicted that small-molecule inhibitors, including heat-shock protein 90, histone deacetylase, PI3K/AKT, and proteasome inhibitors, could reverse the gene expression changes induced by NOTCH1. When tested in vitro, histone deacetylase, PI3K and proteasome inhibitors synergized with GSI in suppressing T-ALL cell growth in GSI-sensitive cells. Interestingly, alvespimycin, a potent inhibitor of the heat-shock protein 90 molecular chaperone, markedly inhibited the growth of both GSI-sensitive and -resistant T-ALL cells, suggesting that its loss disrupts signal transduction pathways crucial for the growth and survival of T-ALL cells.

Presenilin-Based Genetic Screens in Drosophila melanogaster Identify Novel Notch Pathway Modifiers

Presenilin is the enzymatic component of γ-secretase, a multisubunit intramembrane protease that processes several transmembrane receptors, such as the amyloid precursor protein (APP). Mutations in human Presenilins lead to altered APP cleavage and early-onset Alzheimers disease. Presenilins also play an essential role in Notch receptor cleavage and signaling. The Notch pathway is a highly conserved signaling pathway that functions during the development of multicellular organisms, including vertebrates, Drosophila, and C. elegans. Recent studies have shown that Notch signaling is sensitive to perturbations in subcellular trafficking, although the specific mechanisms are largely unknown. To identify genes that regulate Notch pathway function, we have performed two genetic screens in Drosophila for modifiers of Presenilin-dependent Notch phenotypes. We describe here the cloning and identification of 19 modifiers, including nicastrin and several genes with previously undescribed involvement in Notch biology. The predicted functions of these newly identified genes are consistent with extracellular matrix and vesicular trafficking mechanisms in Presenilin and Notch pathway regulation and suggest a novel role for γ-tubulin in the pathway.

De novo Discovery of a γ-Secretase Inhibitor Response Signature Using a Novel In vivo Breast Tumor Model

Notch pathway signaling plays a fundamental role in normal biological processes and is frequently deregulated in many cancers. Although several hypotheses regarding cancer subpopulations most likely to respond to therapies targeting the Notch pathway have been proposed, clinical utility of these predictive markers has not been shown. To understand the molecular basis of gamma-secretase inhibitor (GSI) sensitivity in breast cancer, we undertook an unbiased, de novo responder identification study using a novel genetically engineered in vivo breast cancer model. We show that tumors arising from this model are heterogeneous on the levels of gene expression, histopathology, growth rate, expression of Notch pathway markers, and response to GSI treatment. In addition, GSI treatment of this model was associated with inhibition of Hes1 and proliferation markers, indicating that GSI treatment inhibits Notch signaling. We then identified a pretreatment gene expression signature comprising 768 genes that is significantly associated with in vivo GSI efficacy across 99 tumor lines. Pathway analysis showed that the GSI responder signature is enriched for Notch pathway components and inflammation/immune-related genes. These data show the power of this novel in vivo model system for the discovery of biomarkers predictive of response to targeted therapies, and provide a basis for the identification of human breast cancers most likely to be sensitive to GSI treatment.

Identification of the endosomal sorting complex required for transport-I (ESCRT-I) as an important modulator of anti-miR uptake by cancer cells

Mechanisms of unassisted delivery of RNA therapeutics, including inhibitors of microRNAs, remain poorly understood. We observed that the hepatocellular carcinoma cell line SKHEP1 retains productive free uptake of a miR-21 inhibitor (anti-miR-21). Uptake of anti-miR-21, but not a mismatch (MM) control, induces expression of known miR-21 targets (DDAH1, ANKRD46) and leads to dose-dependent inhibition of cell growth. To elucidate mechanisms of SKHEP1 sensitivity to anti-miR-21, we conducted an unbiased shRNA screen that revealed tumor susceptibility gene 101 (TSG101), a component of the endosomal sorting complex required for transport (ESCRT-I), as an important determinant of anti-proliferative effects of anti-miR-21. RNA interference-mediated knockdown of TSG101 and another ESCRT-I protein, VPS28, improved uptake of anti-miR-21 in parental SKHEP1 cells and restored productive uptake to SKHEP1 clones with acquired resistance to anti-miR-21. Depletion of ESCRT-I in several additional cancer cell lines with inherently poor uptake resulted in improved activity of anti-miR-21. Finally, knockdown of TSG101 increased uptake of anti-miR-21 by cancer cells in vivo following systemic delivery. Collectively, these data support an important role for the ESCRT-I complex in the regulation of productive free uptake of anti-miRs and reveal potential avenues for improving oligonucleotide free uptake by cancer cells.

Glutaminase is essential for the growth of triple-negative breast cancer cells with a deregulated glutamine metabolism pathway and its suppression synergizes with mTOR inhibition

Tumor cells display fundamental changes in metabolism and nutrient uptake in order to utilize additional nutrient sources to meet their enhanced bioenergetic requirements. Glutamine (Gln) is one such nutrient that is rapidly taken up by tumor cells to fulfill this increased metabolic demand. A vital step in the catabolism of glutamine is its conversion to glutamate by the mitochondrial enzyme glutaminase (GLS). This study has identified GLS a potential therapeutic target in breast cancer, specifically in the basal subtype that exhibits a deregulated glutaminolysis pathway. Using inducible shRNA mediated gene knockdown, we discovered that loss of GLS function in triple-negative breast cancer (TNBC) cell lines with a deregulated glutaminolysis pathway led to profound tumor growth inhibition in vitro and in vivo. GLS knockdown had no effect on growth and metabolite levels in non-TNBC cell lines. We rescued the anti-tumor effect of GLS knockdown using shRNA resistant cDNAs encoding both GLS isoforms and by addition of an α-ketoglutarate (αKG) analog thus confirming the critical role of GLS in TNBC. Pharmacological inhibition of GLS with the small molecule inhibitor CB-839 reduced cell growth and led to a decrease in mammalian target of rapamycin (mTOR) activity and an increase in the stress response pathway driven by activating transcription factor 4 (ATF4). Finally, we found that GLS inhibition synergizes with mTOR inhibition, which introduces the possibility of a novel therapeutic strategy for TNBC. Our study revealed that GLS is essential for the survival of TNBC with a deregulated glutaminolysis pathway. The synergistic activity of GLS and mTOR inhibitors in TNBC cell lines suggests therapeutic potential of this combination for the treatment of vulnerable subpopulations of TNBC.

Abstract LB-324: First isoform selective inhibitor of FGFR4 for the treatment of genomically defined patients with hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide. Chemotherapy has proven ineffective, and Sorafenib remains the only approved targeted drug with no second or third line treatment options. Sorafenib slows the growth of advanced liver cancers and helps some patients live longer - by an average of about three months. There is a pressing need for more effective therapies. FGF19 is a highly controlled hormone normally expressed in the intestine, that acts in the liver to regulate bile acid synthesis and hepatocyte proliferation via activation of FGFR4. In 7% of patients with HCC, FGF19 is contained within a focal amplification on chromosome 11q13.3. Overexpression of FGF19 in transgenic mice produces liver tumors which are sensitive to treatment with a FGFR4 tool antibody. Additionally, the growth of tumor cells in xenograft models with FGF19 amplification is dependent on FGFR4 signaling. Thus, selective inhibition of FGFR4 might represent a viable strategy for treating this genetically defined subgroup of HCC patients. Herein, we describe our efforts to identify an ultraselective small molecule inhibitor of FGFR4 which spares the other FGFR isoforms with the intent to avoid FGFR1-3 driven, dose limiting toxicities like soft tissue mineralization. Utilizing structure based drug design, we prepared a series of inhibitor templates designed to covalently modify a target cysteine residue present in FGFR4, but not the other FGFR isoforms. Optimization of one of these templates led to the identification of BLU9931, a highly potent and exquisitely selective, covalent inhibitor of FGFR4. BLU9931 persistently inhibits FGFR4 mediated signaling in cancer cells as evidenced by decreased phosphorylation of FRS2 and ERK. Importantly, BLU9931 does not block signaling driven by FGFR1. Upon oral dosing in mice, BLU9931 is well tolerated and demonstrates robust and dose dependent induction of the FGFR4 target gene CYP7a1 in Hep3B cells, a FGF19 amplified HCC xenograft model. Upon extended dosing, BLU9931 causes sustained regression of tumors, including complete responses. We then explored if HCCs with alterations other than FGF19 amplification are also dependent on FGFR4 signaling. Dosing of molecularly annotated patient derived HCC xenografts with BLU9931 suggests that selective targeting of FGFR4 represents a viable option for the treatment of an expanded population segment of genomically defined HCC patients, much larger than originally anticipated. Citation Format: Margit Hagel, Chandra Miduturu, Mike Sheets, Weifan Weng, Nooreen Rubin, Neil Bifulco, Lucian DiPietro, Joseph Kim, Natasja Brooijmans, Nicolas Stransky, Christopher Winter, Christoph Lengauer, Timothy Guzi. First isoform selective inhibitor of FGFR4 for the treatment of genomically defined patients with hepatocellular carcinoma. [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-324. doi:10.1158/1538-7445.AM2014-LB-324

Oncogenic dependency on β-catenin in liver cancer cell lines correlates with pathway activation

Hepatocellular carcinoma (HCC) represents a serious public health challenge with few therapeutic options available to cancer patients.Wnt/β-catenin pathway is thought to play a significant role in HCC pathogenesis. In this study, we confirmed high frequency of CTNNB1 (β-catenin) mutations in two independent cohorts of HCC patients and demonstrated significant upregulation of β-catenin protein in the overwhelming majority of HCC patient samples, patient-derived xenografts (PDX) and established cell lines. Using genetic tools validated for target specificity through phenotypic rescue experiments, we went on to investigate oncogenic dependency on β-catenin in an extensive collection of human HCC cells lines. Our results demonstrate that dependency on β-catenin generally tracks with its activation status. HCC cell lines that harbored activating mutations in CTNNB1 or displayed elevated levels of non-phosphorylated (active) β-catenin were significantly more sensitive to β-catenin siRNA treatment than cell lines with wild-type CTNNB1 and lower active β-catenin. Finally, significant therapeutic benefit of β-catenin knock-down was demonstrated in established HCC tumor xenografts using doxycycline-inducible shRNA system. β-catenin downregulation and tumor growth inhibition was associated with reduction in AXIN2, direct transcriptional target of β-catenin, and decreased cancer cell proliferation as measured by Ki67 staining. Taken together, our data highlight fundamental importance of aberrant β-catenin signaling in the maintenance of oncogenic phenotype in HCC.

Abstract 1110: Functional genomics reveals genetic dependencies in gastric cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Gastric cancer is the second leading cause of cancer deaths in the world. The genomics of gastric cancers is unique in that they harbor significantly more copy number alterations compared to point mutations, yet the functional importance of these genetic alterations in tumor maintenance is not known. To better understand oncogenic drivers of gastric cancer and identify potential therapeutic targets we performed negative selection RNAi screens in ten well annotated gastric cancer cell lines. Screens were performed using two different but overlapping shRNA libraries. The first library was the Decipher Human Module I pool from Cellecta composed of 27500 shRNAs targeting 5043 genes. The second library was a custom designed focused pool with 6500 shRNAs targeting 608 genes. In addition to screening the two shRNA libraries in vitro, the focused pool was also screened in subcutaneous xenograft tumor models in eight of the gastric cancer cell lines. The screens revealed distinct genetic vulnerabilities that correlated with the corresponding genomic alteration in the specific cell lines. In particular we found that KRAS amplifications confer dependency to the same degree as activating KRAS mutations. This KRAS dependency was further validated with additional shRNAs in KRAS amplified and mutated cell lines. Furthermore, we identified AMPK which is focally amplified in 9% of gastric cancer as a critical oncogenic driver. Multiple subunits of the AMPK holoenzyme scored in the screen and dependency on AMPK alpha and beta subunits was demonstrated with independent shRNAs in two cell lines from the primary screen. Consistent with the screen results we find that LMSU, a gastric cancer cell line not part of the primary screen but annotated as amplified for the AMPK alpha subunit shows elevated expression levels and is sensitive to knockdown of AMPK. These observations have identified AMPK as a novel oncogenic driver in gastric cancer with therapeutic potential. Citation Format: Meghana M. Kulkarni, Sushma Gurumurthy, Oleg Schmidt-Kittler, Jason Berglund, Christopher H. Hulton, David J. Wilson, David Jakubosky, Daniel Michaud, Robert E. Jones, Nicole M. Sjoblom, Russell McSweeney, Hongwei Zhou, Annapurna Venkatakrishnan, Karin J. Jensen, Jingxin Zhang, Parminder K. Mankoo, Jack Pollard, Christopher Winter, Pasi A. Janne, Kwok-Kin Wong, Victoria M. Richon, Jessie M. English, Mark A. Bittinger. Functional genomics reveals genetic dependencies in gastric cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1110. doi:10.1158/1538-7445.AM2015-1110