Amanda J. Redig

Adaptive resistance to therapeutic PD-1 blockade is associated with upregulation of alternative immune checkpoints

Despite compelling antitumour activity of antibodies targeting the programmed death 1 (PD-1): programmed death ligand 1 (PD-L1) immune checkpoint in lung cancer, resistance to these therapies has increasingly been observed. In this study, to elucidate mechanisms of adaptive resistance, we analyse the tumour immune microenvironment in the context of anti-PD-1 therapy in two fully immunocompetent mouse models of lung adenocarcinoma. In tumours progressing following response to anti-PD-1 therapy, we observe upregulation of alternative immune checkpoints, notably T-cell immunoglobulin mucin-3 (TIM-3), in PD-1 antibody bound T cells and demonstrate a survival advantage with addition of a TIM-3 blocking antibody following failure of PD-1 blockade. Two patients who developed adaptive resistance to anti-PD-1 treatment also show a similar TIM-3 upregulation in blocking antibody-bound T cells at treatment failure. These data suggest that upregulation of TIM-3 and other immune checkpoints may be targetable biomarkers associated with adaptive resistance to PD-1 blockade.

Breast cancer as a systemic disease: a view of metastasis.

Breast cancer is now the most frequently diagnosed cancer and leading cause of cancer death in women worldwide. Strategies targeting the primary tumour have markedly improved, but systemic treatments to prevent metastasis are less effective; metastatic disease remains the underlying cause of death in the majority of patients with breast cancer who succumb to their disease. The long latency period between initial treatment and eventual recurrence in some patients suggests that a tumour may both alter and respond to the host systemic environment to facilitate and sustain disease progression. Results from studies in animal models suggest that specific subtypes of breast cancer may direct metastasis through recruitment and activation of haematopoietic cells. In this review, we focus on data implicating breast cancer as a systemic disease.

Basket Trials and the Evolution of Clinical Trial Design in an Era of Genomic Medicine

Since the days of the ancient Greeks, the pathologic hallmarks of malignancy have been reflected in the language of oncology. Hippocrates was the first to use carcinoma—or crab—to describe the familiar invading sweep of tumor cells across tissue planes, and several hundred years later, Galen described the oncos—or swelling—of tumors from which the field of oncology takes its name. However, although the histopathology of malignancy has remained unchanged across several millennia, scientific advances of the modern era have begun to challenge earlier views of oncology, where patients were treated with an exclusive focus on the tissue of origin of a tumor. The translation of next-generation sequencing (NGS) into oncology practice has begun to demonstrate that although the primary site of origin of a tumor matters, so too does its genetic landscape. The significance of classifying tumors based on defining genetic alterations has been particularly relevant for thoracic oncology. The development of tyrosine kinase inhibitors for patients harboring mutations in the epidermal growth factor receptor (EGFR) has been one of the success stories of translational research endeavors and has spurred efforts to identify other driver mutations in lung cancer. National Comprehensive Cancer Network guidelines for non–smallcell lung cancer (NSCLC) now include genetic testing, and the list of potentially actionable mutations continues to grow. However, despite the tremendous promise of this new era of oncology, several challenges have emerged. First, a genetic classification and treatment strategy may not always follow the traditional boundaries of histopathology. Aberrant HER2 signaling is well established in breast cancer but is also an oncogenic driver in a small subset of lung cancers. Similarly, BRAF mutations are most often associated with melanoma but can be found in hairy cell leukemia, colon cancer, lung cancer, thyroid cancer, and brain tumors. Consequently, although we diagnose patients with lung cancer or breast cancer, the genetic makeup of a tumor may be just as important when considering treatment strategies. A lung tumor and a breast tumor with inappropriate activation of the same signaling pathways may share more molecular vulnerabilities with each other than with a lung or breast tumor lacking the same mutations. However, how are such patients to be identified and directed toward appropriate clinical trials? Even the most effective of targeted therapies fail to impress when evaluated in the wrong patient population, as illustrated by early trials with EGFR inhibitors in unselected patients. Furthermore, despite increasing recognition of the importance of genomic analysis in oncology practice, evaluating targeted therapies can present a formidable challenge when the mutations in question are rare and found across disease types. Some eminently targetable mutations may be so rare they are only discovered in the context of a negative trial. In recent reports, the mammalian target of rapamycin inhibitor everolimus caused extraordinary and durable efficacy for two specific patients, despite a lack of efficacy in bladder and thyroid cancers generally. These exceptional responders were retrospectively found to harbor specific mutations in mammalian target of rapamycin signaling pathways, which rendered them uniquely sensitive to everolimus. If other patients with similar mutations can be identified, everolimus may well be the treatment of choice irrespective of tissue diagnosis. As our ability to probe the genome of an individual tumor continues to expand, so too must strategies for clinical trial design. In the article accompanying this editorial, Lopez-Chavez et al present the results of the CUSTOM (Molecular Profiling and Targeted Therapies in Advanced Thoracic Malignancies) trial, a so-called basket trial seeking to identify molecular biomarkers in advanced NSCLC, small-cell lung cancer, and thymic malignancies and to simultaneously evaluate five targeted therapies in patients grouped by molecular markers along with tumor histology. The five targeted therapies included erlotinib (EGFR mutations), the MEK inhibitor selumetinib (KRAS, HRAS, NRAS, and BRAF mutations), the AKT inhibitor MK2206 (PIK3CA, AKT1, and PTEN mutations), lapatinib (HER2 mutations), and sunitinib (KIT and PDGFRA mutations). An attempt was made to evaluate each treatment in all three histologic subtypes, for a total of 15 study arms. Basket trials are a new and evolving form of clinical trial design and are predicated on the hypothesis that the presence of a molecular marker predicts response to a targeted therapy independent of tumor histology. In the study reported by Lopez-Chavez et al, the targeted therapies and actionable mutations were identified prospectively, with patients assigned in a nonrandomized way to a specific treatment arm. The intention of this design is to conduct several independent and parallel phase II trials. Of note, some trials also considered to have a basket design may start with the use of a targeted therapy in an unselected population followed by NGS in patients who respond to identify genetic biomarkers for subsequent prospective screening. Basket trials have generated an enormous amount of interest because JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 33 NUMBER 9 MARCH 2

Biochemical role of the collagen-rich tumour microenvironment in pancreatic cancer progression

PDAC (pancreatic ductal adenocarcinoma) is among the most deadly of human malignances. A hallmark of the disease is a pronounced collagen-rich fibrotic extracellular matrix known as the desmoplastic reaction. Intriguingly, it is precisely these areas of fibrosis in which human PDAC tumours demonstrate increased expression of a key collagenase, MT1-MMP [membrane-type 1 MMP (matrix metalloproteinase); also known as MMP-14]. Furthermore, a cytokine known to mediate fibrosis in vivo, TGF-β1 (transforming growth factor-β1), is up-regulated in human PDAC tumours and can promote MT1-MMP expression. In the present review, we examine the regulation of PDAC progression through the interplay between type I collagen (the most common extracellular matrix present in human PDAC tumours), MT1-MMP and TGF-β1. Specifically, we examine the way in which signalling events through these pathways mediates invasion, regulates microRNAs and contributes to chemoresistance.

Dual mTORC2/mTORC1 targeting results in potent suppressive effects on acute myeloid leukemia (AML) progenitors

Purpose: To determine whether mTORC2 and rapamycin-insensitive (RI)-mTORC1 complexes are present in acute myeloid leukemia (AML) cells and to examine the effects of dual mTORC2/mTORC1 inhibition on primitive AML leukemic progenitors. Experimental Design: Combinations of different experimental approaches were used, including immunoblotting to detect phosphorylated/activated forms of elements of the mTOR pathway in leukemic cell lines and primary AML blasts; cell-proliferation assays; direct assessment of mRNA translation in polysomal fractions of leukemic cells; and clonogenic assays in methylcellulose to evaluate leukemic progenitor-colony formation. Results: mTORC2 complexes are active in AML cells and play critical roles in leukemogenesis. RI-mTORC1 complexes are also formed and regulate the activity of the translational repressor 4E-BP1 in AML cells. OSI-027 blocks mTORC1 and mTORC2 activities and suppresses mRNA translation of cyclin D1 and other genes that mediate proliferative responses in AML cells. Moreover, OSI-027 acts as a potent suppressor of primitive leukemic precursors from AML patients and is much more effective than rapamycin in eliciting antileukemic effects in vitro. Conclusions: Dual targeting of mTORC2 and mTORC1 results in potent suppressive effects on primitive leukemic progenitors from AML patients. Inhibition of the mTOR catalytic site with OSI-027 results in suppression of both mTORC2 and RI-mTORC1 complexes and elicits much more potent antileukemic responses than selective mTORC1 targeting with rapamycin. Clin Cancer Res; 17(13); 4378–88. ©2011 AACR.

Type I interferon (IFN)-dependent activation of Mnk1 and its role in the generation of growth inhibitory responses

We provide evidence for the existence of an IFN-regulated cellular pathway involving the mitogen-activated protein kinase (MAPK)-integrating kinase (Mnk) 1. Our data demonstrate that type I (α, β) IFNs induce phosphorylation/activation of Mnk1, which, in turn, regulates phosphorylation of the eukaryotic initiation factor 4E (eIF4E) on Ser-209. Such Mnk activation depends on upstream engagement of Jak1, and requires downstream activation of the Mek/Erk MAPK pathway. In studies using double Mnk1−/−Mnk2−/− knockout mouse embryonic fibroblasts (MEFs), we found that engagement of Mnk kinases is essential for mRNA translation of the Isg15 and Isg54 genes, suggesting an important role for this pathway in mRNA translation of IFN-stimulated genes (ISGs). Importantly, our data demonstrate that pharmacological inhibition of Mnk kinases or siRNA-mediated knockdown of Mnk1 and Mnk2 results in partial reversal of the suppressive effects of IFNα on normal and leukemic hematopoietic progenitors, establishing a key role for this pathway in the generation of the growth inhibitory effects of type I IFNs. Together, our findings establish that the Mnk/eIF4E kinase pathway is activated in an IFN-inducible manner and plays important roles in mRNA translation for ISGs and generation of IFN-inducible anti-proliferative responses.

Suppressive effects of statins on acute promyelocytic leukemia cells.

The family of statins includes pharmacologic inhibitors of the 3-hydroxy-3-methylglutaryl-CoA reductase that are potent regulators of cholesterol biosynthesis. In addition to their cholesterol-lowering effects, statins inhibit cell proliferation and promote apoptosis of malignant cells in vitro, but their potential therapeutic roles in the treatment of malignancies remain to be defined. We examined the effects of statins on the growth and differentiation of acute myeloid leukemia (AML) cells. Atorvastatin and fluvastatin were found to be potent inducers of cell differentiation and apoptosis of the NB4 acute promyelocytic leukemia (APL) cell line. Such effects correlated with activation of the small G-proteins Rac1/Cdc42 and downstream engagement of the c-Jun NH(2)-terminal kinase kinase pathway, whose function was found to be essential for the generation of proapoptotic responses. Importantly, different statins were found to enhance all-trans-retinoic acid (ATRA)-dependent differentiation of APL blasts and reverse resistance to the antileukemic effects of ATRA. In addition, fluvastatin exhibited growth-inhibitory properties on primary bone marrow-derived leukemic progenitors from patients with AML and enhanced the suppressive effects of ATRA on leukemic progenitor colony formation. Altogether, these studies establish that statins exhibit potent antileukemic properties in vitro and raise the possibility that combinations of statins with ATRA may be an effective approach to overcome the development of ATRA resistance by the leukemic cells.

Regulatory effects of mammalian target of rapamycin-mediated signals in the generation of arsenic trioxide responses

Arsenic trioxide (As2O3) is a potent inducer of apoptosis of leukemic cells in vitro and in vivo, but the mechanisms that mediate such effects are not well understood. We provide evidence that the Akt kinase is phosphorylated/activated during treatment of leukemia cells with As2O3, to regulate downstream engagement of mammalian target of rapamycin (mTOR) and its effectors. Using cells with targeted disruption of both the Akt1 and Akt2 genes, we found that induction of arsenic trioxide-dependent apoptosis is strongly enhanced in the absence of these kinases, suggesting that Akt1/Akt2 are activated in a negative feedback regulatory manner, to control generation of As2O3 responses. Consistent with this, As2O3-dependent pro-apoptotic effects are enhanced in double knock-out cells for both isoforms of the p70 S6 kinase (S6k1/S6k2), a downstream effector of Akt and mTOR. On the other hand, As2O3-dependent induction of apoptosis is diminished in cells with targeted disruption of TSC2, a negative upstream effector of mTOR. In studies using primary hematopoietic progenitors from patients with acute myeloid leukemia, we found that pharmacological inhibition of mTOR enhances the suppressive effects of arsenic trioxide on leukemic progenitor colony formation. Moreover, short interfering RNA-mediated inhibition of expression of the negative downstream effector, translational repressor 4E-BP1, partially reverses the effects of As2O3. Altogether, these data provide evidence for a key regulatory role of the Akt/mTOR pathway in the generation of the effects of As2O3, and suggest that targeting this signaling cascade may provide a novel therapeutic approach to enhance the anti-leukemic properties of As2O3.

Regulation of Arsenic Trioxide-induced Cellular Responses by Mnk1 and Mnk2

Arsenic trioxide (As2O3) is a potent inducer of apoptosis of malignant cells in vitro and in vivo, but the precise mechanisms by which it mediates such effects are not well defined. We provide evidence that As2O3 induces phosphorylation/activation of the MAPK signal-integrating kinases (Mnks) 1 and 2 in leukemia cell lines. Such activation is defective in cells with targeted disruption of the p38α MAPK gene, indicating that it requires upstream engagement of the p38 MAPK pathway. Studies using Mnk1–/– or Mnk2–/–, or double Mnk1–/–Mnk2–/– knock-out cells, establish that activation of Mnk1 and Mnk2 by arsenic trioxide regulates downstream phosphorylation of the eukaryotic initiation factor 4E at Ser-209. Importantly, arsenic-induced apoptosis is enhanced in cells with targeted disruption of the Mnk1 and/or Mnk2 genes, suggesting that these kinases are activated in a negative-feedback regulatory manner, to control generation of arsenic trioxide responses. Consistent with this, pharmacological inhibition of Mnk activity enhances the suppressive effects of arsenic trioxide on primary leukemic progenitors from patients with acute leukemias. Taken together, these findings indicate an important role for Mnk kinases, acting as negative regulators for signals that control generation of arsenic trioxide-dependent apoptosis and antileukemic responses.

Regulation of mammalian target of rapamycin and mitogen activated protein kinase pathways by BCR-ABL.

A large body of evidence has established that BCR–ABL regulates engagement and activation of mammalian target of rapamycin (mTOR) and mitogen activated protein kinase (MAPK) signaling cascades. mTOR-mediated signals, as well as signals transduced by ERK, JNK, and p38 MAPK, are important components of the aberrant signaling induced by BCR–ABL. Such deregulation of mTOR or MAPK pathways contributes to BCR–ABL leukemogenesis, and their targeting with selective inhibitors provides an approach to enhance antileukemic responses and/or overcome leukemic cell resistance in chronic myeloid leukemia (CML) and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). This review explores recent advances in our understanding of mTOR and MAPK signaling in BCR–ABL-expressing leukemias and discusses the potential therapeutic targeting of these pathways in CML and Ph+ ALL.