Faculty Bibliography

Basal levels of autophagy are elevated in most pancreatic ductal adenocarcinomas (PDAC). Suppressing autophagy pharmacologically using chloroquine (CQ) or genetically with RNAi to essential autophagy genes inhibits human pancreatic cancer growth in vitro and in vivo, which presents possible treatment opportunities for PDAC patients using the CQ-derivative hydroxychloroquine (HCQ). Indeed, such clinical trials are ongoing. However, autophagy is a complex cellular mechanism to maintain cell homeostasis under stress. Based on its biological role, a dual role of autophagy in tumorigenesis has been proposed: at tumor initiation, autophagy helps maintain genomic stability and prevent tumor initiation; while in advanced disease, autophagy degrades and recycles cellular components to meet the metabolic needs for rapid growth. This model was proven to be the case in mouse lung tumor models. However, in contrast to prior work in various PDAC model systems, loss of autophagy in PDAC mouse models with embryonic homozygous Trp53 deletion does not inhibit tumor growth and paradoxically increases progression. This raised concerns whether there may be a genotype-dependent reliance of PDAC on autophagy. In a recent study, our group used a Trp53 heterozygous mouse PDAC model and human PDX xenografts to address the question. Our results demonstrate that autophagy inhibition was effective against PDAC tumors irrespective of TP53/TRP53 status.

Pancreatic ductal adenocarcinoma is refractory to available therapies. We have previously shown that these tumors have elevated autophagy and that inhibition of autophagy leads to decreased tumor growth. Using an autochthonous model of pancreatic cancer driven by oncogenic Kras and the stochastic LOH of Trp53, we demonstrate that although genetic ablation of autophagy in the pancreas leads to increased tumor initiation, these premalignant lesions are impaired in their ability to progress to invasive cancer, leading to prolonged survival. In addition, mouse pancreatic cancer cell lines with differing p53 status are all sensitive to pharmacologic and genetic inhibition of autophagy. Finally, a mouse preclinical trial using cohorts of genetically characterized patient-derived xenografts treated with hydroxychloroquine showed responses across the collection of tumors. Together, our data support the critical role of autophagy in pancreatic cancer and show that inhibition of autophagy may have clinical utility in the treatment of these cancers, independent of p53 status. SIGNIFICANCE: Recently, a mouse model with embryonic homozygous Trp53 deletion showed paradoxical effects of autophagy inhibition. We used a mouse model with Trp53 LOH (similar to human tumors), tumor cell lines, and patient-derived xenografts to show that p53 status does not affect response to autophagy inhibition. These findings have important implications on ongoing clinical trials.

Pancreatic ductal adenocarcinomas (PDA) are extremely aggressive cancers and currently available therapies are only minimally effective in treating this disease. Tackling this devastating cancer has been a major challenge to the scientific and medical communities, in part due to its intense therapeutic resistance. One of the aspects of this tumor that contributes to its aggressive behavior is its altered cellular metabolism. Indeed, PDA cells seem to possess the ability to adapt their metabolism to the particular environment to which they are exposed, including utilizing diverse fuel sources depending on their availability. Moreover, PDA tumors are efficient at recycling various metabolic substrates through activation of different salvage pathways such as autophagy and macropinocytosis. Together, these diverse metabolic adaptations allow PDA cells to survive and thrive in harsh environments that may lack nutrients and oxygen. Not surprisingly, given its central role in the pathogenesis of this tumor, oncogenic Kras plays a critical role in much of the metabolic reprogramming seen in PDA. In this review, we discuss the metabolic landscape of PDA tumors, including the molecular underpinnings of the key regulatory nodes, and describe how such pathways can be exploited for future diagnostic and therapeutic approaches.

BACKGROUND: Autophagy is a catabolic pathway that permits cells to recycle intracellular macromolecules, and its inhibition reduces pancreatic cancer growth in model systems. We evaluated hydoxychloroquine (HCQ), an inhibitor of autophagy, in patients with pancreatic cancer and analyzed pharmacodynamic markers in treated patients and mice. METHODS: Patients with previously treated metastatic pancreatic cancer were administered HCQ at 400 mg (n = 10) or 600 mg (n = 10) twice daily. The primary endpoint was 2-month progression-free survival (PFS). We analyzed peripheral lymphocytes from treated mice to identify pharmacodynamic markers of autophagy inhibition that were then assessed in peripheral lymphocytes from patients. RESULTS: Among 20 patients enrolled, 2 (10%) were without progressive disease at 2 months. Median PFS and overall survival were 46.5 and 69.0 days, respectively. Treatment-related grade 3/4 adverse events were lymphopenia (n = 1) and elevated alanine aminotransferase (n = 1). Tolerability and efficacy were similar at the two dose levels. Analysis of treated murine lymphocytes suggested that LC3-II expression by Western blot is a reliable marker for autophagy inhibition. Analysis of LC3-II in patient lymphocytes demonstrated inconsistent autophagy inhibition. CONCLUSION: Mouse studies identified LC3-II levels in peripheral lymphocytes as a potential pharmacodynamic marker of autophagy inhibition. In patients with previously treated metastatic pancreatic cancer, HCQ monotherapy achieved inconsistent autophagy inhibition and demonstrated negligible therapeutic efficacy.

Oncogene-induced DNA damage elicits genomic instability in epithelial cancer cells, but apoptosis is blocked through inactivation of the tumor suppressor p53. In hematological cancers, the relevance of ongoing DNA damage and the mechanisms by which apoptosis is suppressed are largely unknown. We found pervasive DNA damage in hematologic malignancies, including multiple myeloma, lymphoma and leukemia, which leads to activation of a p53-independent, proapoptotic network centered on nuclear relocalization of ABL1 kinase. Although nuclear ABL1 triggers cell death through its interaction with the Hippo pathway coactivator YAP1 in normal cells, we show that low YAP1 levels prevent nuclear ABL1-induced apoptosis in these hematologic malignancies. YAP1 is under the control of a serine-threonine kinase, STK4. Notably, genetic inactivation of STK4 restores YAP1 levels, triggering cell death in vitro and in vivo. Our data therefore identify a new synthetic-lethal strategy to selectively target cancer cells presenting with endogenous DNA damage and low YAP1 levels.

Autophagy, the process by which proteins and organelles are sequestered in double-membrane structures called autophagosomes and delivered to lysosomes for degradation, is critical in diseases such as cancer and neurodegeneration. Much of our understanding of this process has emerged from analysis of bulk cytoplasmic autophagy, but our understanding of how specific cargo, including organelles, proteins or intracellular pathogens, are targeted for selective autophagy is limited. Here we use quantitative proteomics to identify a cohort of novel and known autophagosome-enriched proteins in human cells, including cargo receptors. Like known cargo receptors, nuclear receptor coactivator 4 (NCOA4) was highly enriched in autophagosomes, and associated with ATG8 proteins that recruit cargo-receptor complexes into autophagosomes. Unbiased identification of NCOA4-associated proteins revealed ferritin heavy and light chains, components of an iron-filled cage structure that protects cells from reactive iron species but is degraded via autophagy to release iron through an unknown mechanism. We found that delivery of ferritin to lysosomes required NCOA4, and an inability of NCOA4-deficient cells to degrade ferritin led to decreased bioavailable intracellular iron. This work identifies NCOA4 as a selective cargo receptor for autophagic turnover of ferritin (ferritinophagy), which is critical for iron homeostasis, and provides a resource for further dissection of autophagosomal cargo-receptor connectivity.

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) have been discovered in several cancer types and cause the neurometabolic syndrome D2-hydroxyglutaric aciduria (D2HGA). The mutant enzymes exhibit neomorphic activity resulting in production of D2-hydroxyglutaric acid (D-2HG). To study the pathophysiological consequences of the accumulation of D-2HG, we generated transgenic mice with conditionally activated IDH2(R140Q) and IDH2(R172K) alleles. Global induction of mutant IDH2 expression in adults resulted in dilated cardiomyopathy, white matter abnormalities throughout the central nervous system (CNS), and muscular dystrophy. Embryonic activation of mutant IDH2 resulted in more pronounced phenotypes, including runting, hydrocephalus, and shortened life span, recapitulating the abnormalities observed in D2HGA patients. The diseased hearts exhibited mitochondrial damage and glycogen accumulation with a concordant up-regulation of genes involved in glycogen biosynthesis. Notably, mild cardiac hypertrophy was also observed in nude mice implanted with IDH2(R140Q)-expressing xenografts, suggesting that 2HG may potentially act in a paracrine fashion. Finally, we show that silencing of IDH2(R140Q) in mice with an inducible transgene restores heart function by lowering 2HG levels. Together, these findings indicate that inhibitors of mutant IDH2 may be beneficial in the treatment of D2HGA and suggest that 2HG produced by IDH mutant tumors has the potential to provoke a paraneoplastic condition.

Oncogenic KRAS mutation is the signature genetic event in the progression and growth of pancreatic ductal adenocarcinoma (PDAC), an almost universally fatal disease. Although it has been appreciated for some time that nearly 95% of PDAC harbor mutationally activated KRAS, to date no effective treatments that target this mutant protein have reached the clinic. A number of studies have shown that oncogenic KRAS plays a central role in controlling tumor metabolism by orchestrating multiple metabolic changes including stimulation of glucose uptake, differential channeling of glucose intermediates, reprogrammed glutamine metabolism, increased autophagy, and macropinocytosis. We review these recent findings and address how they may be applied to develop new PDAC treatments.

In early 2011, a dialogue was initiated within the Board of Directors (BOD) of the American Society for Radiation Oncology (ASTRO) regarding the future of the basic sciences of the specialty, primarily focused on the current state and potential future direction of basic research within radiation oncology. After consideration of the complexity of the issues involved and the precise nature of the undertaking, in August 2011, the BOD empanelled a Cancer Biology/Radiation Biology Task Force (TF). The TF was charged with developing an accurate snapshot of the current state of basic (preclinical) research in radiation oncology from the perspective of relevance to the modern clinical practice of radiation oncology as well as the education of our trainees and attending physicians in the biological sciences. The TF was further charged with making suggestions as to critical areas of biological basic research investigation that might be most likely to maintain and build further the scientific foundation and vitality of radiation oncology as an independent and vibrant medical specialty. It was not within the scope of service of the TF to consider the quality of ongoing research efforts within the broader radiation oncology space, to presume to consider their future potential, or to discourage in any way the investigators committed to areas of interest other than those targeted. The TF charge specifically precluded consideration of research issues related to technology, physics, or clinical investigations. This document represents an Executive Summary of the Task Force report.

XBP1 is a critical transcriptional activator of the unfolded protein response (UPR), which increases tumor cell survival under prolonged endoplasmic reticulum (ER) stress and hypoxic conditions.This study was designed to evaluate the immunogenicity of heteroclitic XBP1 unspliced (US)184-192 (YISPWILAV) and heteroclictic XBP1 spliced (SP)367-375 (YLFPQLISV) HLA-A2 peptides, and to characterize the specific activities of XBP1 peptides-specific cytotoxic T lymphocytes (XBP1-CTL) against breast cancer, colon cancer, and pancreatic cancer cells.The XBP1-CTL had upregulated expression of critical T cell markers and displayed HLA-A2-restricted and antigen-specific activities against breast cancer, colon cancer and pancreatic cancer cells. XBP1-CTL were enriched withCD45RO+ memory CTL, which showed high expression of critical T cell markers (CD28, ICOS, CD69, CD40L), cell proliferation and antitumor activities as compared to CD45RO- non-memory CTL. The effector memory (EM: CD45RO+CCR7-) subset had the highest level of cell proliferation while the central memory (CM: CD45RO+CCR7+) subset demonstrated enhanced functional activities (CD107a degranulation, IFNgamma/IL-2 production) upon recognition of the respective tumor cells. Furthermore, both the EM and CM XBP1-CTL subsets expressed high levels of Th1 transcription regulators Tbet and Eomes. The highest frequencies of IFNgamma or granzyme B producing cells were detected within CM XBP1-CTL subset that were either Tbet+ or Eomes+ in responding to the tumor cells.These results demonstrate the immunotherapeutic potential of a cocktail of immunogenic HLA-A2 specific heteroclitic XBP1 US184-192 and heteroclictic XBP1 SP367-375 peptides to induce CD3+CD8+ CTL enriched for CM and EM cells with specific antitumor activities against a variety of solid tumors.