Doctor of Philosophy

dissertationObesity, type 2 diabetes and cancer are serious threats to human health and are increasing at an alarming rate. Dysregulation of energy homeostasis, both at the whole body and cellular level, has been implicated in the pathogenesis of those disorders. Cellular energy homeostasis is a delicate balance between ATP production and consumption. ATP production, mainly from oxidative phosphorylation in mitochondria, is responsive to nutrient availability and cellular energy demand. Therefore, nutrient sensing is critical to maintain cellular energy homeostasis. There are two wellcharacterized nutrient sensing kinases, AMPK and mTOR. We studied another nutrientresponsive kinase named PASK. Using PASKA mice, the role of PASK in cellular energy homeostasis was revealed. PASK deletion caused nearly complete protection from the deleterious effects of a high-fat diet, including obesity, insulin resistance and hepatic steatosis. This protection is likely due to the increased metabolic rate of PASKA mice, which can be recapitulated in cultured cells upon PASK knockdown. Therefore, we conclude that PASK acts as a cell-autonomous metabolic sensor to maintain cellular energy homeostasis. In addition to PASK, we studied a previously uncharacterized but highly conserved mitochondrial protein that we later named Sdh5. Using yeast as primary model system, we showed that Sdh5 is necessary and probably sufficient for insertion of the requisite FAD cofactor into the catalytic subunit of the succinate dehydrogenase (SDH) complex. SDH deficiency has been associated with several types of cancer, predominantly paraganglioma (PGL). Indeed, three out of four familial PGL genes have been mapped to SDH subunits. We discovered a point mutation in the human SDH5 gene that perfectly cosegregates with disease in a Dutch PGL2 lineage. This mutation completely abolishes hSDH5 function and, as expected, PGL2 tumors exhibit a loss of FAD insertion in succinate dehydrogenase. Our studies of PASK and SDH5 not only contribute significant conceptual advances in cellular nutrient sensing and mitochondrial metabolism, but also provides new screening biomarkers and therapeutic targets for diagnosing and treating the metabolic syndrome and cancer

Structure of LFW527

Structure of LFW52

Control of Wnt receptor turnover by R-spondin-ZNRF3/RNF43 signaling module and its dysregulation in cancer

Aberrant activation of the Wnt/β-catenin pathway is frequently found in various cancers, often through mutations of downstream components. Inhibiting β-catenin signaling in tumors with downstream pathway mutations remains challenging, due to a lack of favorable targets. On the other hand, targeting upstream components of the Wnt pathway is rather straightforward. However, it is difficult to identify tumors addicted to autocrine or paracrine Wnt signaling. Discovery of the R-spondin-ZNRF3/RNF43 signaling module and its genetic alterations in cancers represents a breakthrough in this area. Membrane E3 ligase ZNRF3 and RNF43 are critical negative feedback regulators of the Wnt pathway, which function through promoting ubiquitination and degradation of Wnt receptors. R-spondin proteins (RSPO1-4) serve as natural antagonists of ZNRF3/RNF43. To maintain strong and sustained Wnt/β-catenin signaling, cancers need to overcome ZNRF3/RNF43-mediated feedback inhibition. Indeed, mutations of RNF43/ZNRF3 and recurrent translocations of RSPO2/RSPO3 have recently been identified in various cancers. Significantly, genetic alterations in RNF43/ZNRF3/RSPO2/RSPO3 have shown promise as predictive biomarkers in pre-clinical models for the efficacy of upstream Wnt inhibitors. In this review, we will discuss the biology of the R-spondin-ZNRF3/RNF43 signaling module, cancer-associated alterations of this signaling module, and their value as biomarkers to identify Wnt-addicted tumors

Protein phosphatase 1 regulatory subunit 1A regulates cell cycle progression in Ewing sarcoma

Introduction: We recently identified protein phosphatase 1 regulatory subunit 1A (PPP1R1A) as oneof the EWS/FLI core targets that promotes tumor growth and metastasis in Ewing sarcoma (ES), an aggressive pediatric bone and soft tissue tumor. In the current study, we seek to further define the role of PPP1R1A in ES and identify rational combinatorial therapy with improved and specific efficacy in treating primary and metastatic ES. Experimental design: We evaluated ES cell proliferation and cell cycle progression in control and PPP1R1A depleted ES cells. PPP1R1A regulation of cell cycle modulators was analyzed to characterize the underlying mechanism of PPP1R1A mediated cell cycle control. The effects of combination of PPP1R1A and IGF-1R inhibition on ES cell viability and migration in vitro as well as tumor growth and metastasis in an orthotopic xenograft mouse model were investigated. Results: PPP1R1A regulates ES cell cycle in G1/S phase by down-regulating cell cycle inhibitors p21Cip1 and p27Kip1 which results in Rb protein hyperphosphorylation and by promoting normal transcription of replication-dependent histone genes. Furthermore, the combination of PPP1R1A and IGF-1R inhibition induced a synergistic/additive effect on decreasing cell proliferation and migration in vitro and xenograft tumor growth and metastasis in vivo. Conclusions: Taken together, our findings suggest a role of PPP1R1A as an ES specific cell cycle modulator and that simultaneous targeting of PPP1R1A and IGF-1R pathways is a promising specific and effective strategy to treat both primary and metastatic ES

Infigratinib mediates vascular normalization, impairs metastasis and improves chemotherapy in hepatocellular carcinoma.

The fibroblast growth factor (FGF) signaling cascade is a key signaling pathway in hepatocarcinogenesis. We report high FGFR expression in 17.7% (11 of 62) of HCC models. Infigratinib, a pan-FGFR inhibitor, potently suppresses the growth of high-FGFR-expressing and Sorafenib-resistant HCCs. Infigratinib inhibits FGFR signaling and its downstream targets, cell proliferation, the angiogenic rescue program, hypoxia, invasion and metastasis. Infigratinib also induces apoptosis and vessel normalization and improves the overall survival of mice bearing FGFR-driven HCCs. Infigratinib acts in synergy with the microtubule-depolymerizing drug Vinorelbine to promote apoptosis, suppress tumor growth and improve the overall survival of mice. Increased expression levels of FGFR-2 and FGFR-3 via gene amplification correlate with treatment response and may serve as potential biomarkers for patient selection. Conclusion Treatments with Infigratinib alone or in combination with Vinorelbine may be effective in a subset of HCC patients with FGFR-driven tumors

Resistance to allosteric SHP2 inhibition in FGFR-driven cancers through rapid feedback activation of FGFR

SHP2 mediates RAS activation downstream of multiple receptor tyrosine kinases (RTKs) and cancer cell lines dependent on RTKs are in general dependent on SHP2. Profiling of the allosteric SHP2 inhibitor SHP099 across cancer cell lines harboring various RTK dependencies reveals that FGFR-dependent cells are often insensitive to SHP099 when compared to EGFR-dependent cells. We find that FGFR-driven cells depend on SHP2 but exhibit resistance to SHP2 inhibitors in vitro and in vivo. Treatment of such models with SHP2 inhibitors results in an initial decrease in phosphorylated ERK1/2 (p-ERK) levels, however p-ERK levels rapidly rebound within two hours. This p-ERK rebound is blocked by FGFR inhibitors or high doses of SHP2 inhibitors. Mechanistically, compared with EGFR-driven cells, FGFR-driven cells tend to express high levels of RTK negative regulators such as the SPRY family proteins, which are rapidly downregulated upon ERK inhibition. Moreover, over-expression of SPRY4 in FGFR-driven cells prevents MAPK pathway reactivation and sensitizes them to SHP2 inhibitors. We also identified two novel combination approaches to enhance the efficacy of SHP2 inhibitors, either with a distinct site 2 allosteric SHP2 inhibitor or with a RAS-SOS1 interaction inhibitor. Our findings suggest the rapid FGFR feedback activation following initial pathway inhibition by SHP2 inhibitors may promote the open conformation of SHP2 and lead to resistance to SHP2 inhibitors. These findings may assist to refine patient selection and predict resistance mechanisms in the clinical development of SHP2 inhibitors and to suggest strategies for discovering SHP2 inhibitors that are more effective against upstream feedback activation

Capmatinib (INC280) is active against models of non–small cell lung cancer and other cancer types with defined mechanisms of MET activation

Purpose: The selective MET inhibitor capmatinib is being investigated in multiple clinical trials, both as a single agent and in combination. Here, we describe the preclinical data of capmatinib, which supported the clinical biomarker strategy for rational patient selection. Experimental Design: The selectivity and cellular activity of capmatinib were assessed in large cellular screening panels. Antitumor efficacy was quantified in a large set of cell line– or patient-derived xenograft models, testing single-agent or combination treatment depending on the genomic profile of the respective models. Results: Capmatinib was found to be highly selective for MET over other kinases. It was active against cancer models that are characterized by MET amplification, marked MET overexpression, MET exon 14 skipping mutations, or MET activation via expression of the ligand hepatocyte growth factor (HGF). In cancer models where MET is the dominant oncogenic driver, anticancer activity could be further enhanced by combination treatments, for example, by the addition of apoptosis-inducing BH3 mimetics. The combinations of capmatinib and other kinase inhibitors resulted in enhanced anticancer activity against models where MET activation co-occurred with other oncogenic drivers, for example EGFR activating mutations. Conclusions: Activity of capmatinib in preclinical models is associated with a small number of plausible genomic features. The low fraction of cancer models that respond to capmatinib as a single agent suggests that the implementation of patient selection strategies based on these biomarkers is critical for clinical development. Capmatinib is also a rational combination partner for other kinase inhibitors to combat MET-driven resistance

SHP2 inhibition overcomes RTK-mediated pathway re-activation in KRAS mutant tumors treated with MEK inhibitors

FGFR1 was recently shown to be activated as part of a compensatory response to prolonged treatment with MEK inhibitor trametinib in several KRAS mutant lung and pancreatic cancer cell lines. We hypothesize that other receptor tyrosine kinases (RTKs) are also feedback activated in this context. Herein, we profile a large panel of KRAS mutant cancer cell lines for the contribution of RTKs to the feedback activation of phospho-MEK following MEK inhibition, using a SHP2 inhibitor (SHP099) that blocks RAS activation mediated by multiple RTKs. We find that RTK-driven feedback activation widely exists in KRAS mutant cancer cells, to a less extent in those harboring the G13D variant, and involves several RTKs including EGFR, FGFR, and MET. We further demonstrate this pathway feedback activation is mediated through mutant KRAS, at least for the G12C, G12D and G12V variants, and wild-type KRAS can also contribute significantly to the feedback activation. Finally, SHP099 and MEK inhibitors exhibit combination benefits inhibiting KRAS mutant cancer cell proliferation in vitro and in vivo. These findings provide a rationale for exploration of combining SHP2 and MAPK pathway inhibitors for treating KRAS mutant cancers in the clinic

Loss of Tuberous Sclerosis Complex 2 (TSC2) Confers Sensitivity to mTOR Inhibitor Everolimus in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide and hyper-activation of mammalian target of rapamycin (mTOR) signaling plays a pivotal role in HCC tumorigenesis. Tuberous Sclerosis Complex (TSC), a heterodimer of TSC1 and TSC2, functions as a negative regulator of mTOR signaling. In the present study, we discovered that TSC2 loss of function is common in HCC. TSC2 loss was found in 4 of 8 HCC cell lines and 8 of 28 (28.5%) patient-derived HCC xenografts. TSC2 mutations and deletions are likely to be the underlying cause of TSC2 loss in HCC cell lines, xenografts and tumors for most cases. We further demonstrated that TSC2-null HCC cell lines and xenografts have elevated mTOR signaling and, more importantly, were significantly more sensitive to RAD001 (everolimus), an mTOR inhibitor. These preclinical findings led to the analysis of TSC2 status in HCC samples collected in the EVOLVE-1 clinical trial of everolimus. Using an optimized IHC assay we developed, 15 samples with low to undetectable levels of TSC2 (10.8%) were identified (5 in the placebo arm and 10 in the everolimus arm). Although the sample size lacked power to demonstrate statistical significance, TSC2-null/low tumor patients who received everolimus had noticeably longer overall survival than those who received placebo. We also observed that TSC2 loss is rare in Caucasian samples compared to Asian samples. Therefore, we performed an epidemiology survey of more than 239 Asian HCC tumors. The frequency of TSC2 loss is estimated to be around 20% in Asian HBV+ HCC, which accounts for nearly half of HCC malignancies in the world. Taken together, our data strongly argue that TSC2 loss is a predictive biomarker for the sensitivity to everolimus in HCC patients and can be applied as a robust selection biomarker for mTOR inhibitor clinical trials

Combinations with allosteric SHP2 inhibitor TNO155 to block receptor tyrosine kinase signaling

Purpose: SHP2 inhibitors offer an appealing and novel approach to inhibit receptor tyrosine kinase (RTK) signaling, which is the oncogenic driver in many tumors or is frequently feedback activated in response to targeted therapies including RTK inhibitors and MAPK inhibitors. We seek to evaluate the efficacy and synergistic mechanisms of combinations with a novel SHP2 inhibitor, TNO155, to inform their clinical development. Experimental Design: The combinations of TNO155 with EGFR inhibitors (EGFRi), BRAFi, KRASG12Ci, CDK4/6i, and anti–programmed cell death-1 (PD-1) antibody were tested in appropriate cancer models in vitro and in vivo, and their effects on downstream signaling were examined. Results: In EGFR-mutant lung cancer models, combination benefit of TNO155 and the EGFRi nazartinib was observed, coincident with sustained ERK inhibition. In BRAFV600E colorectal cancer models, TNO155 synergized with BRAF plus MEK inhibitors by blocking ERK feedback activation by different RTKs. In KRASG12C cancer cells, TNO155 effectively blocked the feedback activation of wild-type KRAS or other RAS isoforms induced by KRASG12Ci and greatly enhanced efficacy. In addition, TNO155 and the CDK4/6 inhibitor ribociclib showed combination benefit in a large panel of lung and colorectal cancer patient–derived xenografts, including those with KRAS mutations. Finally, TNO155 effectively inhibited RAS activation by colony-stimulating factor 1 receptor, which is critical for the maturation of immunosuppressive tumor-associated macrophages, and showed combination activity with anti–PD-1 antibody. Conclusions: Our findings suggest TNO155 is an effective agent for blocking both tumor-promoting and immune-suppressive RTK signaling in RTK- and MAPK-driven cancers and their tumor microenvironment. Our data provide the rationale for evaluating these combinations clinically