Targeting RSPO3-LGR4 Signaling for Leukemia Stem Cell Eradication in Acute Myeloid Leukemia.

Signals driving aberrant self-renewal in the heterogeneous leukemia stem cell (LSC) pool determine aggressiveness of acute myeloid leukemia (AML). We report that a positive modulator of canonical WNT signaling pathway, RSPO-LGR4, upregulates key self-renewal genes and is essential for LSC self-renewal in a subset of AML. RSPO2/3 serve as stem cell growth factors to block differentiation and promote proliferation of primary AML patient blasts. RSPO receptor, LGR4, is epigenetically upregulated and works through cooperation with HOXA9, a poor prognostic predictor. Blocking the RSPO3-LGR4 interaction by clinical-grade anti-RSPO3 antibody (OMP-131R10/rosmantuzumab) impairs self-renewal and induces differentiation in AML patient-derived xenografts but does not affect normal hematopoietic stem cells, providing a therapeutic opportunity for HOXA9-dependent leukemia.

RSPO3 antagonism inhibits growth and tumorigenicity in colorectal tumors harboring common Wnt pathway mutations.

Activating mutations in the Wnt pathway are a characteristic feature of colorectal cancer (CRC). The R-spondin (RSPO) family is a group of secreted proteins that enhance Wnt signaling and RSPO2 and RSPO3 gene fusions have been reported in CRC. We have previously shown that Wnt pathway blockers exhibit potent combinatorial activity with taxanes to inhibit tumor growth. Here we show that RSPO3 antagonism synergizes with paclitaxel based chemotherapies in patient-derived xenograft models (PDX) with RSPO3 fusions and in tumors with common CRC mutations such as APC, ß-catenin, or RNF43. In these latter types of tumors that represent over 90% of CRC, RSPO3 is produced by stromal cells in the tumor microenvironment and the activating mutations appear to sensitize the tumors to Wnt-Rspo synergy. The combination of RSPO3 inhibition and taxane treatment provides an approach to effectively target oncogenic WNT signaling in a significant number of patients with colorectal and other intestinal cancers.

WNT antagonists exhibit unique combinatorial antitumor activity with taxanes by potentiating mitotic cell death.

The WNT pathway mediates intercellular signaling that regulates cell fate in both normal development and cancer. It is widely appreciated that the WNT pathway is frequently dysregulated in human cancers through a variety of genetic and epigenetic mechanisms. Targets in the WNT pathway are being extensively pursued for the development of new anticancer therapies, and we have advanced two WNT antagonists for clinical development: vantictumab (anti-FZD) and ipafricept (FZD8-Fc). We examined the antitumor efficacy of these WNT antagonists in combination with various chemotherapies in a large set of patient-derived xenograft models. In responsive models, WNT blockade led to profound synergy with taxanes such as paclitaxel, and the combination activity with taxanes was consistently more effective than with other classes of chemotherapy. Taxane monotherapy increased the frequency of cells with active WNT signaling. This selection of WNT-active chemotherapy-resistant tumorigenic cells was prevented by WNT-antagonizing biologics and required sequential dosing of the WNT antagonist followed by the taxane. The WNT antagonists potentiated paclitaxel-mediated mitotic blockade and promoted widespread mitotic cell death. By blocking WNT/ß-catenin signaling before mitotic blockade by paclitaxel, we found that this treatment effectively sensitizes cancer stem cells to taxanes. This combination strategy and treatment regimen has been incorporated into ongoing clinical testing for vantictumab and ipafricept.

Intratumoural heterogeneity generated by Notch signalling promotes small-cell lung cancer.

The Notch signalling pathway mediates cell fate decisions and is tumour suppressive or oncogenic depending on the context. During lung development, Notch pathway activation inhibits the differentiation of precursor cells to a neuroendocrine fate. In small-cell lung cancer, an aggressive neuroendocrine lung cancer, loss-of-function mutations in NOTCH genes and the inhibitory effects of ectopic Notch activation indicate that Notch signalling is tumour suppressive. Here we show that Notch signalling can be both tumour suppressive and pro-tumorigenic in small-cell lung cancer. Endogenous activation of the Notch pathway results in a neuroendocrine to non-neuroendocrine fate switch in 10-50% of tumour cells in a mouse model of small-cell lung cancer and in human tumours. This switch is mediated in part by Rest (also known as Nrsf), a transcriptional repressor that inhibits neuroendocrine gene expression. Non-neuroendocrine Notch-active small-cell lung cancer cells are slow growing, consistent with a tumour-suppressive role for Notch, but these cells are also relatively chemoresistant and provide trophic support to neuroendocrine tumour cells, consistent with a pro-tumorigenic role. Importantly, Notch blockade in combination with chemotherapy suppresses tumour growth and delays relapse in pre-clinical models. Thus, small-cell lung cancer tumours generate their own microenvironment via activation of Notch signalling in a subset of tumour cells, and the presence of these cells may serve as a biomarker for the use of Notch pathway inhibitors in combination with chemotherapy in select patients with small-cell lung cancer.

Genetically engineered mouse models: closing the gap between preclinical data and trial outcomes.

The high failure rate of late-stage human clinical trials, particularly in oncology, predicates the need for improved translation of preclinical data from mouse tumor models into clinical predictions. Genetically engineered mouse models (GEMM) may fulfill this need, because they mimic spontaneous and autochthonous disease progression. Using oncogenic Kras-driven GEMMs of lung and pancreatic adenocarcinoma, we recently showed that these models can closely phenocopy human therapeutic responses to standard-of-care treatment regimens. Here we review the successful preclinical application of such GEMMs, as well as the potential for discovering predictive biomarkers and gaining mechanistic insights into clinical outcomes and drug resistance in human cancers.

Influence of protein transduction domains on intracellular delivery of macromolecules.

As the plasma membrane and blood-brain barrier selectively restrict the entry of most compounds into cells to < 500 Da, delivering macromolecules into cells was, until recently, little more than a goal. However, with significant effort to capitalise on therapeutic targets available in the post-genomic era, novel approaches for delivering therapeutic macromolecules are being rapidly developed. The discovery of small cationic peptides, termed peptide/protein transduction domains or cell-penetrating peptides, which cross biological membranes, has emerged as a venerable Trojan horse to transport large, biologically active molecules, such as peptides, proteins and oligonucleotides, into mammalian cells in vitro, as well as in preclinical models and clinical trials in vivo. This review discusses the implications of peptide/protein transduction domain-mediated delivery of macromolecules and their possible uses as important primary drug delivery agents.

Reperfusion-induced translocation of deltaPKC to cardiac mitochondria prevents pyruvate dehydrogenase reactivation.

Cardiac ischemia and reperfusion are associated with loss in the activity of the mitochondrial enzyme pyruvate dehydrogenase (PDH). Pharmacological stimulation of PDH activity improves recovery in contractile function during reperfusion. Signaling mechanisms that control inhibition and reactivation of PDH during reperfusion were therefore investigated. Using an isolated rat heart model, we observed ischemia-induced PDH inhibition with only partial recovery evident on reperfusion. Translocation of the redox-sensitive delta-isoform of protein kinase C (PKC) to the mitochondria occurred during reperfusion. Inhibition of this process resulted in full recovery of PDH activity. Infusion of the deltaPKC activator H2O2 during normoxic perfusion, to mimic one aspect of cardiac reperfusion, resulted in loss in PDH activity that was largely attributable to translocation of deltaPKC to the mitochondria. Evidence indicates that reperfusion-induced translocation of deltaPKC is associated with phosphorylation of the alphaE1 subunit of PDH. A potential mechanism is provided by in vitro data demonstrating that deltaPKC specifically interacts with and phosphorylates pyruvate dehydrogenase kinase (PDK)2. Importantly, this results in activation of PDK2, an enzyme capable of phosphorylating and inhibiting PDH. Thus, translocation of deltaPKC to the mitochondria during reperfusion likely results in activation of PDK2 and phosphorylation-dependent inhibition of PDH.

Protein kinase Cdelta activation induces apoptosis in response to cardiac ischemia and reperfusion damage: a mechanism involving BAD and the mitochondria.

Heart attacks caused by occlusion of coronary arteries are often treated by mechanical or enzymatic removal of the occlusion and reperfusion of the ischemic heart. It is now recognized that reperfusion per se contributes to myocardial damage, and there is a great interest in identifying the molecular basis of this damage. We recently showed that inhibiting protein kinase Cdelta (PKCdelta) protects the heart from ischemia and reperfusion-induced damage. Here, we demonstrate that PKCdelta activity and mitochondrial translocation at the onset of reperfusion mediates apoptosis by facilitating the accumulation and dephosphorylation of the pro-apoptotic BAD (Bcl-2-associated death promoter), dephosphorylation of Akt, cytochrome c release, PARP (poly(ADP-ribose) polymerase) cleavage, and DNA laddering. Our data suggest that PKCdelta activation has a critical proapoptotic role in cardiac responses following ischemia and reperfusion.

Opposing roles of delta and epsilonPKC in cardiac ischemia and reperfusion: targeting the apoptotic machinery.

Heart attacks, or acute myocardial infarctions (AMI), affect more than one million people in the US every year. The damage that occurs to the heart by AMI is often permanent and as a result, the morbidity and mortality rates of patients that experience AMIs continue to be high. Consequently, AMI patients are at significantly increased risks for future myocardial infarctions, decreased heart function, heart failure, and death [Heart and Stroke statistical update. In American Heart Association (2002) 4]. In this review, we discuss the events that lead to cardiac damage by AMI. Specifically, we discuss the current understanding of the role of ischemic damage vs. reperfusion damage, which is induced by the return of blood, oxygen, and nutrients to the organ. We also discuss the role of apoptosis and necrosis in cardiac damage, the means to protect the heart from damage by ischemia and reperfusion, and the role of protein kinase C in these processes.